Methods and products for increasing the rate of healing of tissue wounds

ABSTRACT

Disclosed are methods for increasing the rate of healing of a tissue wound by administering a composition including a therapeutically effective amount of at least one cationic steroid antimicrobial (CSA). Also disclosed herein are methods of promoting wound healing in a subject in need of such promotion, comprising administering a composition comprising a therapeutically effective amount of at least one CSA. Additionally, disclosed herein are compounds and compositions comprising at least one CSA, or a pharmaceutically acceptable salt thereof, for use in the treatment of a tissue wound. Kits comprising such compositions and instructions on such methods are also contemplated herein.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of U.S. patent applicationSer. No. 13/615,324, filed Sep. 13, 2012, which claims the benefit ofU.S. Provisional Patent Application No. 61/534,194, filed Sep. 13, 2011,the disclosures of which are incorporated herein in their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to compositions and methods for increasing therate of healing of tissue wounds in animals using cationic steroidantimicrobials (CSAs).

2. Relevant Technology

Wound healing is the complex and dynamic process of self-repair torestore cellular structures and tissue layers after an injury hasoccurred. The human wound healing process is divided into three distinctphases: the inflammatory phase, the proliferative phase, and theremodeling phase. The wound healing process may be further broken downbeyond the three broad phases to a complex and coordinated series ofbiochemical events that include chemotaxis, phagocytosis,neocollagenesis, collagen degradation, and collagen remodeling to repairthe damage. In addition, angiogenesis, epithelization, and theproduction of new glycosaminoglycans (GAGs) and proteoglycans are vitalto the wound healing process. The culmination of these biologicalprocesses results in the replacement of normal skin structures withfibroblastic mediated scar tissue.

Prior to the initiation of the inflammatory phase, the clotting cascadebegins to stop blood loss via clotting. Once clotting has begun, varioussoluble factors, including chemokines and cytokines, are released toattract cells to the site of injury. Growth factors, which are cytokinesreleased by platelets, stimulate cells to accelerate their rate ofdivision leading to increased healing rates. Platelets also releaseother proinflammatory factors like serotonin, bradykinin,prostaglandins, prostacyclins, thromboxane, and histamine, which serve anumber of purposes, including to increase cell proliferation andmigration to the area and to cause blood vessels to become dilated andporous.

The inflammation phase can be further broken down into four biochemicalprocesses. First, immediately after a blood vessel is breached, rupturedcell membranes release inflammatory factors like thromboxanes andprostaglandins that result in vasoconstriction to prevent blood loss andto collect inflammatory cells and factors. Second, polymorphonuclearneutrophils (PMNs) arrive at the wound site, phagocytose debris andbacteria. Neutrophils also cleanse the wound by secreting proteases thatbreak down damaged tissue. Third, neutrophils cleanse the injured areavia the phagocytosis of debris, bacteria, neutrophils, damaged cells andtissue, and any other foreign cells or material. Macrophages alsosecrete a number of factors that push wound healing toward the nextphase. Finally, inflammation dies down, fewer inflammatory factors aresecreted, existing ones are broken down, and numbers of neutrophils andmacrophages are reduced at the wound site.

The proliferative phase begins with the arrival of fibroblasts at thewound site, marking the onset of the proliferative phase, whichbiologically begins with angiogenesis. Angiogenesis orneovascularization is the process which occurs concurrently withfibroblast proliferation when endothelial cells migrate to the area ofthe wound. Fibroplasia and granulation begin during angiogenesis as aresult of fibroblast recruitment and extracellular matrix formation.Then collagen and fibronectin are deposited by the fibroblast at thesite of tissue granulation. Once collagen deposition has occurred,epithelialization takes place as a result of the presence ofkeratinocytes arriving at the newly deposited extracellular matrix. Thefinal step in the proliferative phase is contraction, which requires thepresence of neomyoblasts to connect the scar tissue to the surroundinghealthy tissue and to pull the edges of the scar tissue together,restoring tensile strength to the skin surrounding the previouslyinjured area.

A need exists to develop compositions and methods to enhance woundhealing.

BRIEF SUMMARY

Disclosed herein are methods and compositions for treating a wound in asubject by increasing the rate of healing. The increased rate of healingis achieved using a cationic steroidal antimicrobial (CSA) compound. TheCSA compounds include a sterol backbone and a plurality of cationicgroups attached thereto.

The treatment of tissue wounds with CSAs has the surprising andunexpected result of increasing the rate of wound healing. Experimentaldata indicates that the increased rate of wound healing is distinct fromthe known anti-microbial effects of CSAs. When applied to wound sites inliving subjects, the CSA compounds described herein have a profound andsurprising effect on the rate of wound healing. The increased rates ofhealing is in many cases several times faster than healing rates usingtraditional antimicrobials. Studies indicate that this increase in woundhealing is at least in part distinct from antimicrobial benefits. CSAshave been found to increase the rate of healing even in wounds whereantimicrobial load is very low or in cases where infection is not anissue.

The increased rate of healing results in healthier and more naturaltissue formation as compared to treatments with traditionalantimicrobial compounds. The mechanism of action for the increased rateof healing is presently being studied. While the present invention isnot limited to any particular mechanism, it is believed that theincreased rate of tissue healing is caused by increases in fibroblasticmigration and enhanced epithelial growth factors at the wound site.Subjects have also exhibited a significantly reduced sensitivity topain. These benefits are unexpected for CSAs, which were thought to onlybe active against foreign microbes.

In some embodiments, a method is described for increasing the rate ofwound healing in a subject. The method includes (i) providing a tissuetreatment composition including a cationic steroidal anti-microbial(CSA) compound, the CSA compound including a steroidal group and aplurality of cationic groups attached thereto; (ii) identifying asubject in need of accelerated healing of a tissue wound; and (iii)contacting the tissue wound with the tissue treatment composition toincrease the rate of healing thereof.

In some embodiments, the invention relates to a CSA for use in a novelmethod of treating wounds. In particular, the invention relates to apharmaceutically acceptable CSA compound having a sterol backbone and aplurality of cationic groups attached thereto, wherein the CSA compoundis for use in increasing the rate of wound healing in a subject.

In some embodiments, the CSA is a compound of Formula (V) or apharmaceutically acceptable salt thereof:

In some embodiments, the CSA, or a pharmaceutically acceptable saltthereof, is selected from the compound of Formula (I):

In some embodiments, the CSA, or a pharmaceutically acceptable saltthereof, is selected from the compound of Formula (Ia):

In some embodiments rings A, B, C, and D are independently saturated, orare fully or partially unsaturated, provided that at least two of ringsA, B, C, and D are saturated; m, n, p, and q are independently 0 or 1;R₁ through R₄, R₆, R₇, R₁₁, R₁₂, R₁₅, R₁₆, and R₁₈ are independentlyselected from the group consisting of hydrogen, hydroxyl, a substitutedor unsubstituted alkyl, a substituted or unsubstituted hydroxyalkyl, asubstituted or unsubstituted alkyloxyalkyl, a substituted orunsubstituted alkylcarboxyalkyl, a substituted or unsubstitutedalkylaminoalkyl, a substituted or unsubstituted alkylaminoalkylamino, asubstituted or unsubstituted alkylaminoalkylaminoalkylamino, asubstituted or unsubstituted aminoalkyl, a substituted or unsubstitutedaryl, a substituted or unsubstituted arylaminoalkyl, a substituted orunsubstituted haloalkyl, a substituted or unsubstituted alkenyl, asubstituted or unsubstituted alkynyl, oxo, a linking group attached to asecond steroid, a substituted or unsubstituted aminoalkyloxy, asubstituted or unsubstituted aminoalkyloxyalkyl, a substituted orunsubstituted aminoalkylcarboxy, a substituted or unsubstitutedaminoalkylaminocarbonyl, a substituted or unsubstitutedaminoalkylcarboxamido, a substituted or unsubstituteddi(alkyl)aminoalkyl, a substituted or unsubstituted C-carboxyalkyl,H₂N—HC(Q₅)-C(O)—O—, H₂N—HC(Q₅)-C(O)—N(H)—, a substituted orunsubstituted azidoalkyloxy, a substituted or unsubstitutedcyanoalkyloxy, P.G.-HN—HC(Q₅)-C(O)—O—, a substituted or unsubstitutedguanidinoalkyloxy, a substituted or unsubstituted quaternary ammoniumalkylcarboxy, and a substituted or unsubstituted guanidinoalkyl carboxy,where Q₅ is a side chain of any amino acid (including a side chain ofglycine, i.e., H), and P.G. is an amino protecting group; and R₅, R₈,R₉, R₁₀, R₁₃, R₁₄ and R₁₇ are independently deleted when one of rings A,B, C, or D is unsaturated so as to complete the valency of the carbonatom at that site, or R₅, R₈, R₉, R₁₀, R₁₃, and R₁₄ are independentlyselected from the group consisting of hydrogen, hydroxyl, a substitutedor unsubstituted alkyl, a substituted or unsubstituted hydroxyalkyl, asubstituted or unsubstituted alkyloxyalkyl, a substituted orunsubstituted aminoalkyl, a substituted or unsubstituted aryl, asubstituted or unsubstituted haloalkyl, a substituted or unsubstitutedalkenyl, a substituted or unsubstituted alkynyl, oxo, a linking groupattached to a second steroid, a substituted or unsubstitutedaminoalkyloxy, a substituted or unsubstituted aminoalkylcarboxy, asubstituted or unsubstituted aminoalkylaminocarbonyl, a substituted orunsubstituted di(alkyl)aminoalkyl, a substituted or unsubstitutedC-carboxyalkyl, H₂N—HC(Q₅)-C(O)—O—, H₂N—HC(Q₅)-C(O)—N(H)—,azidoalkyloxy, cyanoalkyloxy, P.G.-HN—HC(Q₅)-C(O)—O—, guanidinoalkyloxy,and guanidinoalkylcarboxy, where Q₅ is a side chain of any amino acid,P.G. is an amino protecting group, provided that at least two or threeof R₁₋₄, R₆, R₇, R₁₁, R₁₂, R₁₅, R₁₆, R₁₇, and R₁₈ are independentlyselected from the group consisting of a substituted or unsubstitutedaminoalkyl, a substituted or unsubstituted aminoalkyloxy, a substitutedor unsubstituted alkylcarboxyalkyl, a substituted or unsubstitutedalkylaminoalkylamino, a substituted or unsubstitutedalkylaminoalkylaminoalkylamino, a substituted or unsubstitutedaminoalkylcarboxy, a substituted or unsubstituted arylaminoalkyl, asubstituted or unsubstituted aminoalkyloxyaminoalkylaminocarbonyl, asubstituted or unsubstituted aminoalkylaminocarbonyl, a substituted orunsubstituted aminoalkylcarboxyamido, a quaternary ammoniumalkylcarboxy, a substituted or unsubstituted di(alkyl)aminoalkyl, asubstituted or unsubstituted C-carboxyalkyl, H₂N—HC(Q₅)-C(O)—O—,H₂N—HC(Q₅)-C(O)—N(H)—, azidoalkyloxy, cyanoalkyloxy,P.G.-HN—HC(Q₅)-C(O)—O—, a substituted or unsubstitutedguanidinoalkyloxy, and a substituted or unsubstitutedguanidinoalkylcarboxy.

In some embodiments, R₁ through R₄, R₆, R₇, R₁₁, R₁₂, R₁₅, R₁₆, and R₁₈are independently selected from the group consisting of hydrogen,hydroxyl, a substituted or unsubstituted (C₁-C₁₈) alkyl, a substitutedor unsubstituted (C₁-C₁₈) hydroxyalkyl, a substituted or unsubstituted(C₁-C₁₈) alkyloxy-(C₁-C₁₈) alkyl, a substituted or unsubstituted(C₁-C₁₈) alkylcarboxy-(C₁-C₁₈) alkyl, a substituted or unsubstituted(C₁-C₁₈) alkylamino-(C₁-C₁₈)alkyl, a substituted or unsubstituted(C₁-C₁₈) alkylamino-(C₁-C₁₈) alkylamino, a substituted or unsubstituted(C₁-C₁₈) alkylamino-(C₁-C₁₈) alkylamino-(C₁-C₁₈) alkylamino, asubstituted or unsubstituted (C₁-C₁₈) aminoalkyl, a substituted orunsubstituted aryl, a substituted or unsubstituted arylamino-(C₁-C₁₈)alkyl, a substituted or unsubstituted (C₁-C₁₈) haloalkyl, a substitutedor unsubstituted C₂-C₆ alkenyl, a substituted or unsubstituted C₂-C₆alkynyl, oxo, a linking group attached to a second steroid, asubstituted or unsubstituted (C₁-C₁₈) aminoalkyloxy, a substituted orunsubstituted (C₁-C₁₈) aminoalkyloxy-(C₁-C₁₈) alkyl, a substituted orunsubstituted (C₁-C₁₈) aminoalkylcarboxy, a substituted or unsubstituted(C₁-C₁₈) aminoalkylaminocarbonyl, a substituted or unsubstituted(C₁-C₁₈) aminoalkylcarboxamido, a substituted or unsubstituted di(C₁-C₁₈alkyl)aminoalkyl, a substituted or unsubstituted C-carboxy(C₁-C₁₈)alkyl,H₂N—HC(Q₅)-C(O)—O—, H₂N—HC(Q₅)-C(O)—N(H)—, a substituted orunsubstituted (C₁-C₁₈) azidoalkyloxy, a substituted or unsubstituted(C₁-C₁₈) cyanoalkyloxy, P.G.-HN—HC(Q₅)-C(O)—O—, a substituted orunsubstituted (C₁-C₁₈) guanidinoalkyloxy, a substituted or unsubstituted(C₁-C₁₈) quaternary ammonium alkylcarboxy, and a substituted orunsubstituted (C₁-C₁₈) guanidinoalkyl carboxy, where Q₅ is a side chainof any amino acid (including a side chain of glycine, i.e., H), and P.G.is an amino protecting group; R₅, R₈, R₉, R₁₀, R₁₃, R₁₄ and R₁₇ areindependently deleted when one of rings A, B, C, or D is unsaturated soas to complete the valency of the carbon atom at that site, or R₅, R₈,R₉, R₁₀, R₁₃, and R₁₄ are independently selected from the groupconsisting of hydrogen, hydroxyl, a substituted or unsubstituted(C₁-C₁₈) alkyl, a substituted or unsubstituted (C₁-C₁₈) hydroxyalkyl, asubstituted or unsubstituted (C₁-C₁₈) alkyloxy-(C₁-C₁₈) alkyl, asubstituted or unsubstituted (C₁-C₁₈) aminoalkyl, a substituted orunsubstituted aryl, a substituted or unsubstituted (C₁-C₁₈) haloalkyl, asubstituted or unsubstituted (C₂-C₆) alkenyl, a substituted orunsubstituted (C₂-C₆) alkynyl, oxo, a linking group attached to a secondsteroid, a substituted or unsubstituted (C₁-C₁₈) aminoalkyloxy, asubstituted or unsubstituted (C₁-C₁₈) aminoalkylcarboxy, a substitutedor unsubstituted (C₁-C₁₈) aminoalkylaminocarbonyl, a substituted orunsubstituted di(C₁-C₁₈ alkyl)aminoalkyl, a substituted or unsubstitutedC-carboxy(C₁-C₁₈)alkyl, H₂N—HC(Q₅)-C(O)—O—, H₂N—HC(Q₅)-C(O)—N(H)—, asubstituted or unsubstituted (C₁-C₁₈) azidoalkyloxy, a substituted orunsubstituted (C₁-C₁₈) cyanoalkyloxy, P.G.-HN—HC(Q₅)-C(O)—O—, asubstituted or unsubstituted (C₁-C₁₈) guanidinoalkyloxy, and (C₁-C₁₈)guanidinoalkylcarboxy, where Q₅ is a side chain of any amino acid, andP.G. is an amino protecting group; provided that at least two or threeof R₁₋₄, R₆, R₇, R₁₁, R₁₂, R₁₅, R₁₆, R₁₇, and R₁₈ are independentlyselected from the group consisting of a substituted or unsubstituted(C₁-C₁₈) aminoalkyl, a substituted or unsubstituted (C₁-C₁₈)aminoalkyloxy, a substituted or unsubstituted (C₁-C₁₈)alkylcarboxy-(C₁-C₁₈) alkyl, a substituted or unsubstituted (C₁-C₁₈)alkylamino-(C₁-C₁₈) alkylamino, a substituted or unsubstituted (C₁-C₁₈)alkylamino-(C₁-C₁₈) alkylamino (C₁-C₁₈) alkylamino, a substituted orunsubstituted (C₁-C₁₈) aminoalkylcarboxy, a substituted or unsubstitutedarylamino (C₁-C₁₈) alkyl, a substituted or unsubstituted (C₁-C₁₈)aminoalkyloxy (C₁-C₁₈) aminoalkylaminocarbonyl, a substituted orunsubstituted (C₁-C₁₈) aminoalkylaminocarbonyl, a substituted orunsubstituted (C₁-C₁₈) aminoalkylcarboxyamido, a substituted orunsubstituted (C₁-C₁₈) quaternary ammonium alkylcarboxy, a substitutedor unsubstituted di(C₁-C₁₈ alkyl)aminoalkyl, a substituted orunsubstituted C-carboxy(C₁-C₁₈)alkyl, H₂N—HC(Q₅)-C(O)—O—,H₂N—HC(Q₅)-C(O)—N(H)—, a substituted or unsubstituted (C₁-C₁₈)azidoalkyloxy, a substituted or unsubstituted (C₁-C₁₈) cyanoalkyloxy,P.G.-HN—HC(Q₅)-C(O)—O—, a substituted or unsubstituted (C₁-C₁₈)guanidinoalkyloxy, and a substituted or unsubstituted (C₁-C₁₈)guanidinoalkylcarboxy.

In some embodiments, R₁ through R₄, R₆, R₇, R₁₁, R₁₂, R₁₅, R₁₆, and R₁₈are independently selected from the group consisting of hydrogen,hydroxyl, an unsubstituted (C₁-C₁₈) alkyl, unsubstituted (C₁-C₁₈)hydroxyalkyl, unsubstituted (C₁-C₁₈) alkyloxy-(C₁-C₁₈) alkyl,unsubstituted (C₁-C₁₈) alkylcarboxy-(C₁-C₁₈) alkyl, unsubstituted(C₁-C₁₈) alkylamino-(C₁-C₁₈)alkyl, unsubstituted (C₁-C₁₈)alkylamino-(C₁-C₁₈) alkylamino, (C₁-C₁₈) alkylamino-(C₁-C₁₈)alkylamino-(C₁-C₁₈) alkylamino, an unsubstituted (C₁-C₁₈) aminoalkyl, anunsubstituted aryl, an unsubstituted arylamino-(C₁-C₁₈) alkyl, oxo, anunsubstituted (C₁-C₁₈) aminoalkyloxy, an unsubstituted (C₁-C₁₈)aminoalkyloxy-(C₁-C₁₈) alkyl, an unsubstituted (C₁-C₁₈)aminoalkylcarboxy, (C₁-C₁₈) unsubstituted (C₁-C₁₈)aminoalkylaminocarbonyl, an unsubstituted aminoalkylcarboxamido, anunsubstituted di(C₁-C₁₈ alkyl)aminoalkyl, a substituted or unsubstitutedC-carboxy(C₁-C₁₈)alkyl, unsubstituted (C₁-C₁₈) guanidinoalkyloxy,unsubstituted (C₁-C₁₈) quaternary ammonium alkylcarboxy, andunsubstituted (C₁-C₁₈) guanidinoalkyl carboxy; R₅, R₈, R₉, R₁₀, R₁₃, R₁₄and R₁₇ are independently deleted when one of rings A, B, C, or D isunsaturated so as to complete the valency of the carbon atom at thatsite, or R₅, R₈, R₉, R₁₀, R₁₃, and R₁₄ are independently selected fromthe group consisting of hydrogen, hydroxyl, an unsubstituted (C₁-C₁₈)alkyl, unsubstituted (C₁-C₁₈) hydroxyalkyl, unsubstituted (C₁-C₁₈)alkyloxy-(C₁-C₁₈) alkyl, unsubstituted (C₁-C₁₈) alkylcarboxy-(C₁-C₁₈)alkyl, unsubstituted (C₁-C₁₈) alkylamino-(C₁-C₁₈)alkyl, (C₁-C₁₈)alkylamino-(C₁-C₁₈) alkylamino, unsubstituted (C₁-C₁₈)alkylamino-(C₁-C₁₈) alkylamino-(C₁-C₁₈) alkylamino, an unsubstituted(C₁-C₁₈) aminoalkyl, an unsubstituted aryl, an unsubstitutedarylamino-(C₁-C₁₈) alkyl, oxo, an unsubstituted (C₁-C₁₈) aminoalkyloxy,an unsubstituted (C₁-C₁₈) aminoalkyloxy-(C₁-C₁₈) alkyl, an unsubstituted(C₁-C₁₈) aminoalkylcarboxy, an unsubstituted (C₁-C₁₈)aminoalkylaminocarbonyl, an unsubstituted (C₁-C₁₈)aminoalkylcarboxamido, an unsubstituted (C₁-C₁₈ alkyl)aminoalkyl, asubstituted or unsubstituted C-carboxy(C₁-C₈) alkyl, unsubstituted(C₁-C₁₈) guanidinoalkyloxy, unsubstituted (C₁-C₁₈) quaternary ammoniumalkylcarboxy, and unsubstituted (C₁-C₁₈) guanidinoalkyl carboxy;provided that at least two or three of R₁₋₄, R₆, R₇, R₁₁, R₁₂, R₁₅, R₁₆,R₁₇, and R₁₈ are independently selected from the group consisting ofhydrogen, hydroxyl, an unsubstituted (C₁-C₁₈) alkyl, unsubstituted(C₁-C₁₈) hydroxyalkyl, unsubstituted (C₁-C₁₈) alkyloxy-(C₁-C₁₈) alkyl,unsubstituted (C₁-C₁₈) alkylcarboxy-(C₁-C₁₈) alkyl, unsubstituted(C₁-C₁₈) alkylamino-(C₁-C₁₈) alkyl, unsubstituted (C₁-C₁₈)alkylamino-(C₁-C₁₈) alkylamino, unsubstituted (C₁-C₁₈)alkylamino-(C₁-C₁₈) alkylamino-(C₁-C₁₈) alkylamino, an unsubstituted(C₁-C₁₈) aminoalkyl, an unsubstituted aryl, an unsubstitutedarylamino-(C₁-C₁₈) alkyl, oxo, an unsubstituted (C₁-C₁₈) aminoalkyloxy,an unsubstituted (C₁-C₁₈) aminoalkyloxy-(C₁-C₁₈) alkyl, an unsubstituted(C₁-C₁₈) aminoalkylcarboxy, an unsubstituted (C₁-C₁₈)aminoalkylaminocarbonyl, an unsubstituted (C₁-C₁₈)aminoalkyl-carboxamido, an unsubstituted (C₁-C₁₈ alkyl)aminoalkyl, asubstituted or unsubstituted C-carboxy(C₁-C₁₈) alkyl, unsubstituted(C₁-C₁₈) guanidinoalkyloxy, unsubstituted (C₁-C₁₈) quaternary ammoniumalkylcarboxy, and unsubstituted (C₁-C₁₈) guanidinoalkyl carboxy.

In some embodiments, R₃, R₇, R₁₂, and R₁₈ are independently selectedfrom the group consisting of hydrogen, an unsubstituted (C₁-C₁₈) alkyl,unsubstituted (C₁-C₁₈) hydroxyalkyl, unsubstituted (C₁-C₁₈)alkyloxy-(C₁-C₁₈) alkyl, unsubstituted (C₁-C₁₈) alkylcarboxy-(C₁-C₁₈)alkyl, unsubstituted (C₁-C₁₈) alkylamino-(C₁-C₁₈)alkyl, unsubstituted(C₁-C₁₈) alkylamino-(C₁-C₁₈) alkylamino, unsubstituted (C₁-C₁₈)alkylamino-(C₁-C₁₈) alkylamino-(C₁-C₁₈) alkylamino, an unsubstituted(C₁-C₁₈) aminoalkyl, an unsubstituted arylamino-(C₁-C₁₈) alkyl, anunsubstituted (C₁-C₁₈) aminoalkyloxy, an unsubstituted (C₁-C₁₈)aminoalkyloxy-(C₁-C₁₈) alkyl, an unsubstituted (C₁-C₁₈)aminoalkylcarboxy, an unsubstituted (C₁-C₁₈) aminoalkylaminocarbonyl, anunsubstituted (C₁-C₁₈) aminoalkylcarboxamido, an unsubstituted (C₁-C₁₈alkyl)aminoalkyl, a substituted or unsubstituted C-carboxy(C₁-C₁₈)alkyl, unsubstituted (C₁-C₁₈) guanidinoalkyloxy, unsubstituted (C₁-C₁₈)quaternary ammonium alkylcarboxy, and unsubstituted (C₁-C₁₈)guanidinoalkyl carboxy; and R₁, R₂, R₄, R₅, R₆, R₈, R₉, R₁₀, R₁₁, R₁₃,R₁₄, R₁₅, R₁₆, and R₁₇ are independently selected from the groupconsisting of hydrogen and unsubstituted (C₁-C₆) alkyl.

In some embodiments, R₃, R₇, R₁₂, and R₁₈ are independently selectedfrom the group consisting of hydrogen, an unsubstituted (C₁-C₆) alkyl,unsubstituted (C₁-C₆) hydroxyalkyl, unsubstituted (C₁-C₁₆)alkyloxy-(C₁-C₅) alkyl, unsubstituted (C₁-C₁₆) alkylcarboxy-(C₁-C₅)alkyl, unsubstituted (C₁-C₁₆) alkylamino-(C₁-C₅)alkyl, (C₁-C₁₆)alkylamino-(C₁-C₅) alkylamino, unsubstituted (C₁-C₁₆)alkylamino-(C₁-C₁₆) alkylamino-(C₁-C₅) alkylamino, an unsubstituted(C₁-C₁₆) aminoalkyl, an unsubstituted arylamino-(C₁-C₅) alkyl, anunsubstituted (C₁-C₅) aminoalkyloxy, an unsubstituted (C₁-C₁₆)aminoalkyloxy-(C₁-C₅) alkyl, an unsubstituted (C₁-C₅) aminoalkylcarboxy,an unsubstituted (C₁-C₅) aminoalkylaminocarbonyl, an unsubstituted(C₁-C₅) aminoalkylcarboxamido, an unsubstituted di(C₁-C₅alkyl)amino-(C₁-C₅) alkyl, a substituted or unsubstitutedC-carboxy(C₁-C₅)alkyl, unsubstituted (C₁-C₅) guanidinoalkyloxy,unsubstituted (C₁-C₁₆) quaternary ammonium alkylcarboxy, andunsubstituted (C₁-C₁₆) guanidinoalkylcarboxy;

In some embodiments, R₁, R₂, R₄, R₅, R₆, R₈, R₁₀, R₁₁, R₁₄, R₁₆, and R₁₇are each hydrogen; and R₉ and R₁₃ are each methyl.

In some embodiments, R₃, R₇, R₁₂, and R₁₈ are independently selectedfrom the group consisting of aminoalkyloxy; aminoalkylcarboxy;alkylaminoalkyl; alkoxycarbonylalkyl; alkylcarbonylalkyl;di(alkyl)aminoalkyl; C-carboxyalkyl; alkoxycarbonylalkyl; andalkylcarboxyalkyl.

In some embodiments, R₃, R₇, and R₁₂ are independently selected from thegroup consisting of aminoalkyloxy and aminoalkylcarboxy; and R₁₈ isselected from the group consisting of alkylaminoalkyl;alkoxycarbonylalkyl; alkylcarbonyloxyalkyl; di(alkyl)aminoalkyl;C-carboxyalkyl; alkylaminoalkyl; alkyoxycarbonylalkyl; andalkylcarboxyalkyl.

In some embodiments, R₃, R₇, and R₁₂ are the same. In some embodiments,R₃, R₇, and R₁₂ are aminoalkyloxy. In some embodiments, R₃, R₇, and R₁₂are aminoalkylcarboxy.

In some embodiments, R₁₈ is alkylaminoalkyl. In some embodiments,alkoxycarbonylalkyl. In some embodiments, R₁₈ is di(alkyl)aminoalkyl. Insome embodiments, R₁₈ is alkylcarboxyalkyl. In some embodiments, R₁₈ isC-carboxyalkyl.

In some embodiments, R₃, R₇, R₁₂, and R₁₈ are independently selectedfrom the group consisting of amino-C₃-alkyloxy; amino-C₃-alkyl-carboxy;C₈-alkylamino-C₅-alkyl; C₈-alkoxy-carbonyl-C₄-alkyl;C₈-alkyl-carbonyl-C₄-alkyl; di-(C₅-alkyl)amino-C₅-alkyl;C-carboxy-C₄-alkyl; C₁₃-alkylamino-C₅-alkyl;C₆-alkoxy-carbonyl-C₄-alkyl; and C₆-alkyl-carboxy-C₄-alkyl.

In some embodiments, the CSA, or a pharmaceutically acceptable saltthereof, is:

In some embodiments, the CSA, or a pharmaceutically acceptable saltthereof, is

In some embodiments, the pharmaceutically acceptable salt is ahydrochloride salt. In some embodiments, the pharmaceutically acceptablesalt is a tri-hydrochloride salt.

The present invention may be embodied in other specific forms withoutdeparting from its spirit or essential characteristics. The describedembodiments are to be considered in all respects only as illustrativeand not restrictive. The scope of the invention is, therefore, indicatedby the appended claims rather than by the foregoing description. Allchanges which come within the meaning and range of equivalency of theclaims are to be embraced within their scope.

DETAILED DESCRIPTION

The embodiments disclosed herein will now be described by reference tosome more detailed embodiments, with occasional reference to theaccompanying drawings. These embodiments may, however, be embodied indifferent forms and should not be construed as limited to theembodiments set forth herein. Rather, these embodiments are provided sothat this disclosure will be thorough and complete, and will fullyconvey the scope of the embodiments to those skilled in the art.

DEFINITIONS

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which these embodiments belong. The terminology used in thedescription herein is for describing particular embodiments only and isnot intended to be limiting of the embodiments. As used in thespecification and the appended claims, the singular forms “a,” “an,” and“the” are intended to include the plural forms as well, unless thecontext clearly indicates otherwise. All publications, patentapplications, patents, and other references mentioned herein areincorporated by reference in their entirety.

Terms and phrases used in this application, and variations thereof,especially in the appended claims, unless otherwise expressly stated,should be construed as open ended as opposed to limiting. As examples ofthe foregoing, the term ‘including’ should be read to mean ‘including,without limitation,’ ‘including but not limited to,’ or the like; theterm ‘comprising’ as used herein is synonymous with ‘including,’‘containing,’ or ‘characterized by,’ and is inclusive or open-ended anddoes not exclude additional, unrecited elements or method steps; theterm ‘having’ should be interpreted as ‘having at least;’ the term‘includes’ should be interpreted as ‘includes but is not limited to;’the term ‘example’ is used to provide exemplary instances of the item indiscussion, not an exhaustive or limiting list thereof; and use of termslike ‘preferably,’ ‘preferred,’ ‘desired,’ or ‘desirable,’ and words ofsimilar meaning should not be understood as implying that certainfeatures are critical, essential, or even important to the structure orfunction of the invention, but instead as merely intended to highlightalternative or additional features that may or may not be utilized in aparticular embodiment. In addition, the term “comprising” is to beinterpreted synonymously with the phrases “having at least” or“including at least”. When used in the context of a process, the term“comprising” means that the process includes at least the recited steps,but may include additional steps. When used in the context of acompound, composition or device, the term “comprising” means that thecompound, composition or device includes at least the recited featuresor components, but may also include additional features or components.Likewise, a group of items linked with the conjunction ‘and’ should notbe read as requiring that each and every one of those items be presentin the grouping, but rather should be read as ‘and/or’ unless expresslystated otherwise. Similarly, a group of items linked with theconjunction ‘or’ should not be read as requiring mutual exclusivityamong that group, but rather should be read as ‘and/or’ unless expresslystated otherwise.

It is understood that, in any compound described herein having one ormore chiral centers, if an absolute stereochemistry is not expresslyindicated, then each center may independently be of R-configuration orS-configuration or a mixture thereof. Thus, the compounds providedherein may be enantiomerically pure, enantiomerically enriched, racemicmixture, diastereomerically pure, diastereomerically enriched, or astereoisomeric mixture. In addition it is understood that, in anycompound described herein having one or more double bond(s) generatinggeometrical isomers that can be defined as E or Z, each double bond mayindependently be E or Z a mixture thereof.

Likewise, it is understood that, in any compound described, alltautomeric forms are also intended to be included.

It is to be understood that where compounds disclosed herein haveunfilled valencies, then the valencies are to be filled with hydrogensor isotopes thereof, e.g., hydrogen-1 (protium) and hydrogen-2(deuterium).

It is understood that the compounds described herein can be labeledisotopically. Substitution with isotopes such as deuterium may affordcertain therapeutic advantages resulting from greater metabolicstability, such as, for example, increased in vivo half-life or reduceddosage requirements. Each chemical element as represented in a compoundstructure may include any isotope of said element. For example, in acompound structure a hydrogen atom may be explicitly disclosed orunderstood to be present in the compound. At any position of thecompound that a hydrogen atom may be present, the hydrogen atom can beany isotope of hydrogen, including but not limited to hydrogen-1(protium) and hydrogen-2 (deuterium). Thus, reference herein to acompound encompasses all potential isotopic forms unless the contextclearly dictates otherwise.

It is understood that the methods and combinations described hereininclude crystalline forms (also known as polymorphs, which include thedifferent crystal packing arrangements of the same elemental compositionof a compound), amorphous phases, salts, solvates, and hydrates. In someembodiments, the compounds described herein exist in solvated forms withpharmaceutically acceptable solvents such as water, ethanol, or thelike. In other embodiments, the compounds described herein exist inunsolvated form. Solvates contain either stoichiometric ornon-stoichiometric amounts of a solvent, and may be formed during theprocess of crystallization with pharmaceutically acceptable solventssuch as water, ethanol, or the like. Hydrates are formed when thesolvent is water, or alcoholates are formed when the solvent is alcohol.In addition, the compounds provided herein can exist in unsolvated aswell as solvated forms. In general, the solvated forms are consideredequivalent to the unsolvated forms for the purposes of the compounds andmethods provided herein.

Unless otherwise indicated, all numbers expressing quantities ofingredients, reaction conditions, and so forth used in the specificationand claims are to be understood as being modified in all instances bythe term “about.” Accordingly, unless indicated to the contrary, thenumerical parameters set forth in the specification and attached claimsare approximations that may vary depending upon the desired propertiessought to be obtained by the present embodiments. At the very least, andnot as an attempt to limit the application of the doctrine ofequivalents to the scope of the claims, each numerical parameter shouldbe construed in light of the number of significant digits and ordinaryrounding approaches.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the embodiments are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. Any numerical value, however, inherently contains certainerrors necessarily resulting from the standard deviation found in theirrespective testing measurements. Every numerical range given throughoutthis specification and claims will include every narrower numericalrange that falls within such broader numerical range, as if suchnarrower numerical ranges were all expressly written herein. Where arange of values is provided, it is understood that the upper and lowerlimit, and each intervening value between the upper and lower limit ofthe range is encompassed within the embodiments.

As used herein, any “R” group(s) such as, without limitation, R₁, R₂,R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁, R₁₂, R₁₃, R₁₄, R₁₅, R₁₆, R₁₇, andR₁₈ represent substituents that can be attached to the indicated atom.Unless otherwise specified, an R group may be substituted orunsubstituted.

A “ring” as used herein can be heterocyclic or carbocyclic. The term“saturated” used herein refers to a ring having each atom in the ringeither hydrogenated or substituted such that the valency of each atom isfilled. The term “unsaturated” used herein refers to a ring where thevalency of each atom of the ring may not be filled with hydrogen orother substituents. For example, adjacent carbon atoms in the fused ringcan be doubly bound to each other. Unsaturation can also includedeleting at least one of the following pairs and completing the valencyof the ring carbon atoms at these deleted positions with a double bond;such as R₅ and R₉ R₈ and R₁₀; and R₁₃ and R₁₄.

Whenever a group is described as being “substituted” that group may besubstituted with one, two, three or more of the indicated substituents,which may be the same or different, each replacing a hydrogen atom. Ifno substituents are indicated, it is meant that the indicated“substituted” group may be substituted with one or more group(s)individually and independently selected from alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, cycloalkynyl, acylalkyl, alkoxyalkyl,aminoalkyl, amino acid, aryl, heteroaryl, heteroalicyclyl, aralkyl,heteroaralkyl, (heteroalicyclyl)alkyl, hydroxy, protected hydroxyl,alkoxy, aryloxy, acyl, mercapto, alkylthio, arylthio, cyano, halogen(e.g., F, Cl, Br, and I), thiocarbonyl, O-carbamyl, N-carbamyl,O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, S-sulfonamido,N-sulfonamido, C-carboxy, protected C-carboxy, O-carboxy, isocyanato,thiocyanato, isothiocyanato, nitro, oxo, silyl, sulfenyl, sulfinyl,sulfonyl, haloalkyl, haloalkoxy, trihalomethanesulfonyl,trihalomethanesulfonamido, an amino, a mono-substituted amino group anda di-substituted amino group, R_(a)O(CH₂)_(m)O—, R_(b)(CH₂)_(n)O—,R_(c)C(O)O(CH₂)_(p)O—, and protected derivatives thereof. Thesubstituent may be attached to the group at more than one attachmentpoint. For example, an aryl group may be substituted with a heteroarylgroup at two attachment points to form a fused multicyclic aromatic ringsystem. Biphenyl and naphthalene are two examples of an aryl group thatis substituted with a second aryl group.

As used herein, “C_(a)” or “C_(a) to C_(b)” in which “a” and “b” areintegers refer to the number of carbon atoms in an alkyl, alkenyl oralkynyl group, or the number of carbon atoms in the ring of acycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl orheteroalicyclyl group. That is, the alkyl, alkenyl, alkynyl, ring of thecycloalkyl, ring of the cycloalkenyl, ring of the cycloalkynyl, ring ofthe aryl, ring of the heteroaryl or ring of the heteroalicyclyl cancontain from “a” to “b”, inclusive, carbon atoms. Thus, for example, a“C₁ to C₄ alkyl” group refers to all alkyl groups having from 1 to 4carbons, that is, CH₃—, CH₃CH₂—, CH₃CH₂CH₂—, (CH₃)₂CH—, CH₃CH₂CH₂CH₂—,CH₃CH₂CH(CH₃)— and (CH₃)₃C—. If no “a” and “b” are designated withregard to an alkyl, alkenyl, alkynyl, cycloalkyl cycloalkenyl,cycloalkynyl, aryl, heteroaryl or heteroalicyclyl group, the broadestrange described in these definitions is to be assumed.

As used herein, “alkyl” refers to a straight or branched hydrocarbonchain that comprises a fully saturated (no double or triple bonds)hydrocarbon group. The alkyl group may have 1 to 25 carbon atoms(whenever it appears herein, a numerical range such as “1 to 25” refersto each integer in the given range; e.g., “1 to 25 carbon atoms” meansthat the alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3carbon atoms, etc., up to and including 25 carbon atoms, although thepresent definition also covers the occurrence of the term “alkyl” whereno numerical range is designated). The alkyl group may also be a mediumsize alkyl having 1 to 15 carbon atoms. The alkyl group could also be alower alkyl having 1 to 6 carbon atoms. The alkyl group of the compoundsmay be designated as “C₄” or “C₁-C₄ alkyl” or similar designations. Byway of example only, “C₁-C₄ alkyl” indicates that there are one to fourcarbon atoms in the alkyl chain, i.e., the alkyl chain is selected frommethyl, ethyl, propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, andt-butyl. Typical alkyl groups include, but are in no way limited to,methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl,pentyl and hexyl. The alkyl group may be substituted or unsubstituted.

As used herein, “alkenyl” refers to an alkyl group that contains in thestraight or branched hydrocarbon chain one or more double bonds. Thealkenyl group may have 2 to 25 carbon atoms (whenever it appears herein,a numerical range such as “2 to 25” refers to each integer in the givenrange; e.g., “2 to 25 carbon atoms” means that the alkenyl group mayconsist of 2 carbon atom, 3 carbon atoms, 4 carbon atoms, etc., up toand including 25 carbon atoms, although the present definition alsocovers the occurrence of the term “alkenyl” where no numerical range isdesignated). The alkenyl group may also be a medium size alkenyl having2 to 15 carbon atoms. The alkenyl group could also be a lower alkenylhaving 1 to 6 carbon atoms. The alkenyl group of the compounds may bedesignated as “C₄” or “C₂-C₄ alkyl” or similar designations. An alkenylgroup may be unsubstituted or substituted.

As used herein, “alkynyl” refers to an alkyl group that contains in thestraight or branched hydrocarbon chain one or more triple bonds. Thealkynyl group may have 2 to 25 carbon atoms (whenever it appears herein,a numerical range such as “2 to 25” refers to each integer in the givenrange; e.g., “2 to 25 carbon atoms” means that the alkynyl group mayconsist of 2 carbon atom, 3 carbon atoms, 4 carbon atoms, etc., up toand including 25 carbon atoms, although the present definition alsocovers the occurrence of the term “alkynyl” where no numerical range isdesignated). The alkynyl group may also be a medium size alkynyl having2 to 15 carbon atoms. The alkynyl group could also be a lower alkynylhaving 2 to 6 carbon atoms. The alkynyl group of the compounds may bedesignated as “C₄” or “C₂-C₄ alkyl” or similar designations. An alkynylgroup may be unsubstituted or substituted.

As used herein, “aryl” refers to a carbocyclic (all carbon) monocyclicor multicyclic aromatic ring system (including fused ring systems wheretwo carbocyclic rings share a chemical bond) that has a fullydelocalized pi-electron system throughout all the rings. The number ofcarbon atoms in an aryl group can vary. For example, the aryl group canbe a C₆-C₁₄ aryl group, a C₆-C₁₀ aryl group, or a C₆ aryl group(although the definition of C₆-C₁₀ aryl covers the occurrence of “aryl”when no numerical range is designated). Examples of aryl groups include,but are not limited to, benzene, naphthalene and azulene. An aryl groupmay be substituted or unsubstituted.

As used herein, “aralkyl” and “aryl(alkyl)” refer to an aryl groupconnected, as a substituent, via a lower alkylene group. The aralkylgroup may have 6 to 20 carbon atoms (whenever it appears herein, anumerical range such as “6 to 20” refers to each integer in the givenrange; e.g., “6 to 20 carbon atoms” means that the aralkyl group mayconsist of 6 carbon atom, 7 carbon atoms, 8 carbon atoms, etc., up toand including 20 carbon atoms, although the present definition alsocovers the occurrence of the term “aralkyl” where no numerical range isdesignated). The lower alkylene and aryl group of an aralkyl may besubstituted or unsubstituted. Examples include but are not limited tobenzyl, 2-phenylalkyl, 3-phenylalkyl, and naphthylalkyl.

“Lower alkylene groups” refer to a C₁-C₂₅ straight-chained alkyltethering groups, such as —CH₂— tethering groups, forming bonds toconnect molecular fragments via their terminal carbon atoms. Examplesinclude but are not limited to methylene (—CH₂—), ethylene (—CH₂CH₂—),propylene (—CH₂CH₂CH₂—), and butylene (—CH₂CH₂CH₂CH₂—). A lower alkylenegroup can be substituted by replacing one or more hydrogen of the loweralkylene group with a substituent(s) listed under the definition of“substituted.”

As used herein, “cycloalkyl” refers to a completely saturated (no doubleor triple bonds) mono- or multi-cyclic hydrocarbon ring system. Whencomposed of two or more rings, the rings may be joined together in afused fashion. Cycloalkyl groups can contain 3 to 10 atoms in thering(s) or 3 to 8 atoms in the ring(s). A cycloalkyl group may beunsubstituted or substituted. Typical cycloalkyl groups include, but arein no way limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl and cyclooctyl.

As used herein, “cycloalkenyl” refers to a mono- or multi-cyclichydrocarbon ring system that contains one or more double bonds in atleast one ring; although, if there is more than one, the double bondscannot form a fully delocalized pi-electron system throughout all therings (otherwise the group would be “aryl,” as defined herein). Whencomposed of two or more rings, the rings may be connected together in afused fashion. A cycloalkenyl group may be unsubstituted or substituted.

As used herein, “cycloalkynyl” refers to a mono- or multi-cyclichydrocarbon ring system that contains one or more triple bonds in atleast one ring. If there is more than one triple bond, the triple bondscannot form a fully delocalized pi-electron system throughout all therings. When composed of two or more rings, the rings may be joinedtogether in a fused fashion. A cycloalkynyl group may be unsubstitutedor substituted.

As used herein, “alkoxy” or “alkyloxy” refers to the formula —OR whereinR is an alkyl, an alkenyl, an alkynyl, a cycloalkyl, a cycloalkenyl or acycloalkynyl as defined above. A non-limiting list of alkoxys aremethoxy, ethoxy, n-propoxy, 1-methylethoxy (isopropoxy), n-butoxy,iso-butoxy, sec-butoxy and tert-butoxy. An alkoxy may be substituted orunsubstituted.

As used herein, “acyl” refers to a hydrogen, alkyl, alkenyl, alkynyl,aryl, or heteroaryl connected, as substituents, via a carbonyl group.Examples include formyl, acetyl, propanoyl, benzoyl, and acryl. An acylmay be substituted or unsubstituted.

As used herein, “alkoxyalkyl” or “alkyloxyalkyl” refers to an alkoxygroup connected, as a substituent, via a lower alkylene group. Examplesinclude alkyl-O-alkyl- and alkoxy-alkyl- with the terms alkyl and alkoxydefined herein.

As used herein, “hydroxyalkyl” refers to an alkyl group in which one ormore of the hydrogen atoms are replaced by a hydroxy group. Exemplaryhydroxyalkyl groups include but are not limited to, 2-hydroxyethyl,3-hydroxypropyl, 2-hydroxypropyl, and 2,2-dihydroxyethyl. A hydroxyalkylmay be substituted or unsubstituted.

As used herein, “haloalkyl” refers to an alkyl group in which one ormore of the hydrogen atoms are replaced by a halogen (e.g.,mono-haloalkyl, di-haloalkyl and tri-haloalkyl). Such groups include butare not limited to, chloromethyl, fluoromethyl, difluoromethyl,trifluoromethyl and 1-chloro-2-fluoromethyl, 2-fluoroisobutyl. Ahaloalkyl may be substituted or unsubstituted.

The term “amino” as used herein refers to a —NH₂ group.

As used herein, the term “hydroxy” refers to a —OH group.

A “cyano” group refers to a “—CN” group.

A “carbonyl” or an “oxo” group refers to a C═O group.

The term “azido” as used herein refers to a —N₃ group.

As used herein, “aminoalkyl” refers to an amino group connected, as asubstituent, via a lower alkylene group. Examples include H₂N-alkyl-with the term alkyl defined herein.

As used herein, “alkylcarboxyalkyl” refers to an alkyl group connected,as a substituent, to a carboxy group that is connected, as asubstituent, to an alkyl group. Examples include alkyl-C(═O)O-alkyl- andalkyl-O—C(═O)-alkyl- with the term alkyl as defined herein.

As used herein, “C-carboxyalkyl” refers to a carboxy group connected, asa substituent, to an alkyl group. Examples include HO—(C═O)-alkyl, withthe term alkyl as defined herein.

As used herein, “alkylaminoalkyl” refers to an alkyl group connected, asa substituent, to an amino group that is connected, as a substituent, toan alkyl group. Examples include alkyl-NH-alkyl-, with the term alkyl asdefined herein.

As used herein, “dialkylaminoalkyl” or “di(alkyl)aminoalkyl” refers totwo alkyl groups connected, each as a substituent, to an amino groupthat is connected, as a substituent, to an alkyl group. Examples include

with the term alkyl as defined herein.

As used herein, “alkylaminoalkylamino” refers to an alkyl groupconnected, as a substituent, to an amino group that is connected, as asubstituent, to an alkyl group that is connected, as a substituent, toan amino group. Examples include alkyl-NH-alkyl-NH—, with the term alkylas defined herein.

As used herein, “alkylaminoalkylaminoalkylamino” refers to an alkylgroup connected, as a substituent, to an amino group that is connected,as a substituent, to an alkyl group that is connected, as a substituent,to an amino group that is connected, as a substituent, to an alkylgroup. Examples include alkyl-NH-alkyl-NH-alkyl-, with the term alkyl asdefined herein.

As used herein, “arylaminoalkyl” refers to an aryl group connected, as asubstituent, to an amino group that is connected, as a substituent, toan alkyl group. Examples include aryl-NH-alkyl-, with the terms aryl andalkyl as defined herein.

As used herein, “aminoalkyloxy” refers to an amino group connected, as asubstituent, to an alkyloxy group. Examples include H₂N-alkyl-O— andH₂N-alkoxy- with the terms alkyl and alkoxy as defined herein.

As used herein, “aminoalkyloxyalkyl” refers to an amino group connected,as a substituent, to an alkyloxy group connected, as a substituent, toan alkyl group. Examples include H₂N-alkyl-O-alkyl- andH₂N-alkoxy-alkyl- with the terms alkyl and alkoxy as defined herein.

As used herein, “aminoalkylcarboxy” refers to an amino group connected,as a substituent, to an alkyl group connected, as a substituent, to acarboxy group. Examples include H₂N-alkyl-C(═O)O— and H₂N-alkyl-O—C(═O)—with the term alkyl as defined herein.

As used herein, “aminoalkylaminocarbonyl” refers to an amino groupconnected, as a substituent, to an alkyl group connected, as asubstituent, to an amino group connected, as a substituent, to acarbonyl group. Examples include H₂N-alkyl-NH—C(═O)— with the term alkylas defined herein.

As used herein, “aminoalkylcarboxamido” refers to an amino groupconnected, as a substituent, to an alkyl group connected, as asubstituent, to a carbonyl group connected, as a substituent to an aminogroup. Examples include H₂N-alkyl-C(═O)—NH— with the term alkyl asdefined herein.

As used herein, “azidoalkyloxy” refers to an azido group connected as asubstituent, to an alkyloxy group. Examples include N₃-alkyl-O— andN₃-alkoxy- with the terms alkyl and alkoxy as defined herein.

As used herein, “cyanoalkyloxy” refers to a cyano group connected as asubstituent, to an alkyloxy group. Examples include NC-alkyl-O— andNC-alkoxy- with the terms alkyl and alkoxy as defined herein.

As used herein, “guanidinoalkyloxy” refers to a guanidinyl groupconnected, as a substituent, to an alkyloxy group. Examples include

with the terms alkyl and alkoxy as defined herein.

As used herein, “guanidinoalkylcarboxy” refers to a guanidinyl groupconnected, as a substituent, to an alkyl group connected, as asubstituent, to a carboxy group. Examples include

with the term alkyl as defined herein.

As used herein, “quaternary ammonium alkylcarboxy” refers to aquaternized amino group connected, as a substituent, to an alkyl groupconnected, as a substituent, to a carboxy group. Examples include

with the term alkyl as defined herein.

The term “halogen atom” or “halogen” as used herein, means any one ofthe radio-stable atoms of column 7 of the Periodic Table of theElements, such as, fluorine, chlorine, bromine and iodine.

Where the numbers of substituents is not specified (e.g. haloalkyl),there may be one or more substituents present. For example “haloalkyl”may include one or more of the same or different halogens.

As used herein, the term “amino acid” refers to any amino acid (bothstandard and non-standard amino acids), including, but not limited to,α-amino acids, β-amino acids, γ-amino acids and δ-amino acids. Examplesof suitable amino acids include, but are not limited to, alanine,asparagine, aspartate, cysteine, glutamate, glutamine, glycine, proline,serine, tyrosine, arginine, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, threonine, tryptophan and valine. Additionalexamples of suitable amino acids include, but are not limited to,ornithine, hypusine, 2-aminoisobutyric acid, dehydroalanine,gamma-aminobutyric acid, citrulline, beta-alanine, alpha-ethyl-glycine,alpha-propyl-glycine and norleucine.

A linking group is a divalent moiety used to link one steroid to anothersteroid. In some embodiments, the linking group is used to link a firstCSA with a second CSA (which may be the same or different). An exampleof a linking group is (C₁-C₁₀) alkyloxy-(C₁-C₁₀) alkyl.

The terms “P.G.” or “protecting group” or “protecting groups” as usedherein refer to any atom or group of atoms that is added to a moleculein order to prevent existing groups in the molecule from undergoingunwanted chemical reactions. Examples of protecting group moieties aredescribed in T. W. Greene and P. G. M. Wuts, Protective Groups inOrganic Synthesis, 3. Ed. John Wiley & Sons, 1999, and in J. F. W.McOmie, Protective Groups in Organic Chemistry Plenum Press, 1973, bothof which are hereby incorporated by reference for the limited purpose ofdisclosing suitable protecting groups. The protecting group moiety maybe chosen in such a way, that they are stable to certain reactionconditions and readily removed at a convenient stage using methodologyknown from the art. A non-limiting list of protecting groups includebenzyl; substituted benzyl; alkylcarbonyls and alkoxycarbonyls (e.g.,t-butoxycarbonyl (BOC), acetyl, or isobutyryl); arylalkylcarbonyls andarylalkoxycarbonyls (e.g., benzyloxycarbonyl); substituted methyl ether(e.g. methoxymethyl ether); substituted ethyl ether; a substitutedbenzyl ether; tetrahydropyranyl ether; silyls (e.g., trimethylsilyl,triethylsilyl, triisopropylsilyl, t-butyldimethylsilyl,tri-iso-propylsilyloxymethyl, [2-(trimethylsilyl)ethoxy]methyl ort-butyldiphenylsilyl); esters (e.g. benzoate ester); carbonates (e.g.methoxymethylcarbonate); sulfonates (e.g. tosylate or mesylate); acyclicketal (e.g. dimethyl acetal); cyclic ketals (e.g., 1,3-dioxane,1,3-dioxolanes, and those described herein); acyclic acetal; cyclicacetal (e.g., those described herein); acyclic hemiacetal; cyclichemiacetal; cyclic dithioketals (e.g., 1,3-dithiane or 1,3-dithiolane);orthoesters (e.g., those described herein) and triarylmethyl groups(e.g., trityl; monomethoxytrityl (MMTr); 4,4′-dimethoxytrityl (DMTr);4,4′,4″-trimethoxytrityl (TMTr); and those described herein).Amino-protecting groups are known to those skilled in the art. Ingeneral, the species of protecting group is not critical, provided thatit is stable to the conditions of any subsequent reaction(s) on otherpositions of the compound and can be removed at the appropriate pointwithout adversely affecting the remainder of the molecule. In addition,a protecting group may be substituted for another after substantivesynthetic transformations are complete. Clearly, where a compounddiffers from a compound disclosed herein only in that one or moreprotecting groups of the disclosed compound has been substituted with adifferent protecting group, that compound is within the disclosure.

CSA Compounds

Compounds useful in accordance with this disclosure are describedherein, both generically and with particularity, and in U.S. Pat. Nos.6,350,738, 6,486,148, 6,767,904, 7,598,234, 7,754,705, and 8,975,310,which are incorporated herein by reference. Compounds include steroidderivatives, such as cationic steroid antimicrobials (“CSAs”) thatexhibit one or more wound healing activities or functions.

CSA compounds, are synthetically produced small molecules that include asterol backbone having various charged groups (e.g., amine and cationicgroups) attached to the backbone. The backbone can be used to orient theamine or guanidine groups on one face, or plane, of the sterol backbone.

CSAs are cationic and amphiphilic, based upon the functional groupsattached to the backbone. They are facially amphiphilic with ahydrophobic face and a polycationic face. For example, a scheme showinga compound having primary amino groups on one face, or plane, of abackbone is shown below in Scheme I:

The charged groups are believed to be responsible for antimicrobialproperties. For example, the charged groups may disrupt the bacterialcellular membrane to cause cell death or sensitization.

In some embodiments the CSA compound may have a formula as set forth inFormula (V) or a pharmaceutically acceptable salt thereof:

Where m, n, p, and q are independently 0 or 1; R₁-R₁₈ representsubstituents that are attached to the indicated atom on the steroidbackbone (i.e., steroid group); and at least two, preferably at leastthree, of R₁-R₁₈ each include a cationic group.

In one embodiment, rings A, B, C, and D are independently saturated, orare fully or partially unsaturated, provided that at least two of ringsA, B, C, and D are saturated; m, n, p, and q are independently 0 or 1;R₁ through R₄, R₆, R₇, R₁₁, R₁₂, R₁₅, R₁₆, and R₁₈ are independentlyselected from the group consisting of hydrogen, hydroxyl, a substitutedor unsubstituted alkyl, substituted or unsubstituted hydroxyalkyl,substituted or unsubstituted alkyloxyalkyl, substituted or unsubstitutedalkylcarboxyalkyl, substituted or unsubstituted alkylaminoalkyl,substituted or unsubstituted alkylaminoalkylamino, substituted orunsubstituted alkylaminoalkylaminoalkylamino, a substituted orunsubstituted aminoalkyl, a substituted or unsubstituted aryl, asubstituted or unsubstituted arylaminoalkyl, substituted orunsubstituted haloalkyl, substituted or unsubstituted alkenyl,substituted or unsubstituted alkynyl, oxo, a linking group attached to asecond steroid, a substituted or unsubstituted aminoalkyloxy, asubstituted or unsubstituted aminoalkyloxyalkyl, a substituted orunsubstituted aminoalkylcarboxy, a substituted or unsubstitutedaminoalkylaminocarbonyl, a substituted or unsubstitutedaminoalkylcarboxamido, a substituted or unsubstituteddi(alkyl)aminoalkyl, a substituted or unsubstituted C-carboxyalkyl,H₂N—HC(Q₅)-C(O)—O—, H₂N—HC(Q₅)-C(O)—N(H)—, substituted or unsubstitutedazidoalkyloxy, substituted or unsubstituted cyanoalkyloxy,P.G.-HN—HC(Q₅)-C(O)—O—, substituted or unsubstituted guanidinoalkyloxy,substituted or unsubstituted quaternary ammonium alkylcarboxy, andsubstituted or unsubstituted guanidinoalkyl carboxy, where Q₅ is a sidechain of any amino acid (including a side chain of glycine, i.e., H),and P.G. is an amino protecting group; and R₅, R₈, R₉, R₁₀, R₁₃, R₁₄ andR₁₇ are independently deleted when one of rings A, B, C, or D isunsaturated so as to complete the valency of the carbon atom at thatsite, or R₅, R₈, R₉, R₁₀, R₁₃, and R₁₄ are independently selected fromthe group consisting of hydrogen, hydroxyl, a substituted orunsubstituted alkyl, substituted or unsubstituted hydroxyalkyl,substituted or unsubstituted alkyloxyalkyl, a substituted orunsubstituted aminoalkyl, a substituted or unsubstituted aryl,substituted or unsubstituted haloalkyl, substituted or unsubstitutedalkenyl, substituted or unsubstituted alkynyl, oxo, a linking groupattached to a second steroid, a substituted or unsubstitutedaminoalkyloxy, a substituted or unsubstituted aminoalkylcarboxy, asubstituted or unsubstituted aminoalkylaminocarbonyl, a substituted orunsubstituted di(alkyl)aminoalkyl, a substituted or unsubstitutedC-carboxyalkyl, H₂NHC(Q₅)-C(O)—O—, H₂N—HC(Q₅)-C(O)—N(H)—, substituted orunsubstituted azidoalkyloxy, substituted or unsubstituted cyanoalkyloxy,P.G.-HN—HC(Q₅)-C(O)—O—, substituted or unsubstituted guanidinoalkyloxy,and substituted or unsubstituted guanidinoalkylcarboxy, where Q₅ is aside chain of any amino acid, P.G. is an amino protecting group;provided that at least two or three of R₁₋₄, R₆, R₇, R₁₁, R₁₂, R₁₅, R₁₆,R₁₇, and R₁₈ are independently selected from the group consisting of asubstituted or unsubstituted aminoalkyl, a substituted or unsubstitutedaminoalkyloxy, substituted or unsubstituted alkylcarboxyalkyl,substituted or unsubstituted alkylaminoalkylamino, substituted orunsubstituted alkylaminoalkylaminoalkylamino, a substituted orunsubstituted aminoalkylcarboxy, a substituted or unsubstitutedarylaminoalkyl, a substituted or unsubstitutedaminoalkyloxyaminoalkylaminocarbonyl, a substituted or unsubstitutedaminoalkylaminocarbonyl, a substituted or unsubstitutedaminoalkylcarboxyamido, a substituted or unsubstituted quaternaryammonium alkylcarboxy, a substituted or unsubstituteddi(alkyl)aminoalkyl, a substituted or unsubstituted C-carboxyalkyl,H₂N—HC(Q₅)-C(O)—O—, H₂N—HC(Q₅)-C(O)—N(H)—, substituted or unsubstitutedazidoalkyloxy, substituted or unsubstituted cyanoalkyloxy,P.G.-HN—HC(Q₅)-C(O)—O—, substituted or unsubstituted guanidinoalkyloxy,and a substituted or unsubstituted guanidinoalkylcarboxy.

In some embodiments, R₁ through R₄, R₆, R₇, R₁₁, R₁₂, R₁₅, R₁₆, and R₁₈are independently selected from the group consisting of hydrogen,hydroxyl, a substituted or unsubstituted (C₁-C₁₈) alkyl, substituted orunsubstituted (C₁-C₁₈) hydroxyalkyl, substituted or unsubstituted(C₁-C₁₈) alkyloxy-(C₁-C₁₈) alkyl, substituted or unsubstituted (C₁-C₁₈)alkylcarboxy-(C₁-C₁₈) alkyl, substituted or unsubstituted (C₁-C₁₈)alkylamino-(C₁-C₁₈)alkyl, substituted or unsubstituted (C₁-C₁₈)alkylamino-(C₁-C₁₈) alkylamino, substituted or unsubstituted (C₁-C₁₈)alkylamino-(C₁-C₁₈) alkylamino-(C₁-C₁₈) alkylamino, a substituted orunsubstituted (C₁-C₁₈) aminoalkyl, a substituted or unsubstituted aryl,a substituted or unsubstituted arylamino-(C₁-C₁₈) alkyl, substituted orunsubstituted (C₁-C₁₈) haloalkyl, substituted or unsubstituted (C₂-C₆)alkenyl, substituted or unsubstituted (C₂-C₆) alkynyl, oxo, a linkinggroup attached to a second steroid, a substituted or unsubstituted(C₁-C₁₈) aminoalkyloxy, a substituted or unsubstituted (C₁-C₁₈)aminoalkyloxy-(C₁-C₁₈) alkyl, a substituted or unsubstituted (C₁-C₁₈)aminoalkylcarboxy, a substituted or unsubstituted (C₁-C₁₈)aminoalkylaminocarbonyl, a substituted or unsubstituted (C₁-C₁₈)aminoalkylcarboxamido, a substituted or unsubstituted di(C₁-C₁₈alkyl)aminoalkyl, a substituted or unsubstituted C-carboxy(C₁-C₁₈)alkyl,H₂N—HC(Q₅)-C(O)—O—, H₂N—HC(Q₅)-C(O)—N(H)—, substituted or unsubstituted(C₁-C₁₈) azidoalkyloxy, substituted or unsubstituted (C₁-C₁₈)cyanoalkyloxy, P.G.-HN—HC(Q₅)-C(O)—O—, substituted or unsubstituted(C₁-C₁₈) guanidinoalkyloxy, substituted or unsubstituted (C₁-C₁₈)quaternary ammonium alkylcarboxy, and substituted or unsubstituted(C₁-C₁₈) guanidinoalkyl carboxy, where Q₅ is a side chain of any aminoacid (including a side chain of glycine, i.e., H), and P.G. is an aminoprotecting group; and R₅, R₈, R₉, R₁₀, R₁₃, R₁₄ and R₁₇ areindependently deleted when one of rings A, B, C, or D is unsaturated soas to complete the valency of the carbon atom at that site, or R₅, R₈,R₉, R₁₀, R₁₃, and R₁₄ are independently selected from the groupconsisting of hydrogen, hydroxyl, a substituted or unsubstituted(C₁-C₁₈) alkyl, substituted or unsubstituted (C₁-C₁₈) hydroxyalkyl,substituted or unsubstituted (C₁-C₁₈) alkyloxy-(C₁-C₁₈) alkyl, asubstituted or unsubstituted (C₁-C₁₈) aminoalkyl, a substituted orunsubstituted aryl, substituted or unsubstituted (C₁-C₁₈) haloalkyl,substituted or unsubstituted (C₂-C₆) alkenyl, substituted orunsubstituted (C₂-C₆) alkynyl, oxo, a linking group attached to a secondsteroid, a substituted or unsubstituted (C₁-C₁₈) aminoalkyloxy, asubstituted or unsubstituted (C₁-C₁₈) aminoalkylcarboxy, a substitutedor unsubstituted (C₁-C₁₈) aminoalkylaminocarbonyl, di(C₁-C₁₈alkyl)aminoalkyl, a substituted or unsubstituted C-carboxy(C₁-C₁₈)alkyl,H₂N—HC(Q₅)-C(O)—O—, H₂N—HC(Q₅)-C(O)—N(H)—, substituted or unsubstituted(C₁-C₁₈) azidoalkyloxy, substituted or unsubstituted (C₁-C₁₈)cyanoalkyloxy, P.G.-HN—HC(Q₅)-C(O)—O—, substituted or unsubstituted(C₁-C₁₈) guanidinoalkyloxy, and substituted or unsubstituted (C₁-C₁₈)guanidinoalkylcarboxy, where Q₅ is a side chain of any amino acid, andP.G. is an amino protecting group; provided that at least two or threeof R₁₋₄, R₆, R₇, R₁₁, R₁₂, R₁₅, R₁₆, R₁₇, and R₁₈ are independentlyselected from the group consisting of a substituted or unsubstituted(C₁-C₁₈) aminoalkyl, a substituted or unsubstituted (C₁-C₁₈)aminoalkyloxy, substituted or unsubstituted (C₁-C₁₈)alkylcarboxy-(C₁-C₁₈) alkyl, substituted or unsubstituted (C₁-C₁₈)alkylamino-(C₁-C₁₈) alkylamino, substituted or unsubstituted (C₁-C₁₈)alkylamino-(C₁-C₁₈) alkylamino (C₁-C₁₈) alkylamino, a substituted orunsubstituted (C₁-C₁₈) aminoalkylcarboxy, a substituted or unsubstitutedarylamino (C₁-C₁₈) alkyl, a substituted or unsubstituted (C₁-C₁₈)aminoalkyloxy (C₁-C₁₈) aminoalkylaminocarbonyl, a substituted orunsubstituted (C₁-C₁₈) aminoalkylaminocarbonyl, a substituted orunsubstituted (C₁-C₁₈) aminoalkylcarboxyamido, a substituted orunsubstituted (C₁-C₁₈) quaternary ammonium alkylcarboxy, substituted orunsubstituted di(C₁-C₁₈ alkyl)aminoalkyl, a substituted or unsubstitutedC-carboxy(C₁-C₁₈)alkyl, H₂N—HC(Q₅)-C(O)—O—, H₂N—HC(Q₅)-C(O)—N(H)—,substituted or unsubstituted (C₁-C₁₈) azidoalkyloxy, substituted orunsubstituted (C₁-C₁₈) cyanoalkyloxy, P.G.-HN—HC(Q₅)-C(O)—O—,substituted or unsubstituted (C₁-C₁₈) guanidinoalkyloxy, and asubstituted or unsubstituted (C₁-C₁₈) guanidinoalkylcarboxy.

In some embodiments, R₁ through R₄, R₆, R₇, R₁₁, R₁₂, R₁₅, R₁₆, and R₁₈are independently selected from the group consisting of hydrogen,hydroxyl, an unsubstituted (C₁-C₁₈) alkyl, unsubstituted (C₁-C₁₈)hydroxyalkyl, unsubstituted (C₁-C₁₈) alkyloxy-(C₁-C₁₈) alkyl,unsubstituted (C₁-C₁₈) alkylcarboxy-(C₁-C₁₈) alkyl, unsubstituted(C₁-C₁₈) alkylamino-(C₁-C₁₈)alkyl, unsubstituted (C₁-C₁₈)alkylamino-(C₁-C₁₈) alkylamino, unsubstituted (C₁-C₁₈)alkylamino-(C₁-C₁₈) alkylamino-(C₁-C₁₈) alkylamino, an unsubstituted(C₁-C₁₈) aminoalkyl, an unsubstituted aryl, an unsubstitutedarylamino-(C₁-C₁₈) alkyl, oxo, an unsubstituted (C₁-C₁₈) aminoalkyloxy,an unsubstituted (C₁-C₁₈) aminoalkyloxy-(C₁-C₁₈) alkyl, an unsubstituted(C₁-C₁₈) aminoalkylcarboxy, an unsubstituted (C₁-C₁₈)aminoalkylaminocarbonyl, an unsubstituted (C₁-C₁₈)aminoalkylcarboxamido, an unsubstituted di(C₁-C₁₈ alkyl)aminoalkyl, anunsubstituted C-carboxy(C₁-C₁₈)alkyl, unsubstituted (C₁-C₁₈)guanidinoalkyloxy, unsubstituted (C₁-C₁₈) quaternary ammoniumalkylcarboxy, and unsubstituted (C₁-C₁₈) guanidinoalkyl carboxy; and R₅,R₈, R₉, R₁₀, R₁₃, R₁₄ and R₁₇ are independently deleted when one ofrings A, B, C, or D is unsaturated so as to complete the valency of thecarbon atom at that site, or R₅, R₈, R₉, R₁₀, R₁₃, and R₁₄ areindependently selected from the group consisting of hydrogen, hydroxyl,an unsubstituted (C₁-C₁₈) alkyl, unsubstituted (C₁-C₁₈) hydroxyalkyl,unsubstituted (C₁-C₁₈) alkyloxy-(C₁-C₁₈) alkyl, unsubstituted (C₁-C₁₈)alkylcarboxy-(C₁-C₁₈) alkyl, unsubstituted (C₁-C₁₈)alkylamino-(C₁-C₁₈)alkyl, unsubstituted (C₁-C₁₈) alkylamino-(C₁-C₁₈)alkylamino, unsubstituted (C₁-C₁₈) alkylamino-(C₁-C₁₈)alkylamino-(C₁-C₁₈) alkylamino, an unsubstituted (C₁-C₁₈) aminoalkyl, anunsubstituted aryl, an unsubstituted arylamino-(C₁-C₁₈) alkyl, oxo, anunsubstituted (C₁-C₁₈) aminoalkyloxy, an unsubstituted (C₁-C₁₈)aminoalkyloxy-(C₁-C₁₈) alkyl, an unsubstituted (C₁-C₁₈)aminoalkylcarboxy, an unsubstituted (C₁-C₁₈) aminoalkylaminocarbonyl, anunsubstituted (C₁-C₁₈) aminoalkylcarboxamido, an unsubstituted di(C₁-C₁₈alkyl)aminoalkyl, an unsubstituted C-carboxy(C₁-C₁₈)alkyl, unsubstituted(C₁-C₁₈) guanidinoalkyloxy, unsubstituted (C₁-C₁₈) quaternary ammoniumalkylcarboxy, and unsubstituted (C₁-C₁₈) guanidinoalkyl carboxy;provided that at least two or three of R₁₋₄, R₆, R₇, R₁₁, R₁₂, R₁₅, R₁₆,R₁₇, and R₁₈ are independently selected from the group consisting ofhydrogen, hydroxyl, an unsubstituted (C₁-C₁₈) alkyl, unsubstituted(C₁-C₁₈) hydroxyalkyl, unsubstituted (C₁-C₁₈) alkyloxy-(C₁-C₁₈) alkyl,unsubstituted (C₁-C₁₈) alkylcarboxy-(C₁-C₁₈) alkyl, unsubstituted(C₁-C₁₈) alkylamino-(C₁-C₁₈)alkyl, unsubstituted (C₁-C₁₈)alkylamino-(C₁-C₁₈) alkylamino, unsubstituted (C₁-C₁₈)alkylamino-(C₁-C₁₈) alkylamino-(C₁-C₁₈) alkylamino, an unsubstituted(C₁-C₁₈) aminoalkyl, an unsubstituted aryl, an unsubstitutedarylamino-(C₁-C₁₈) alkyl, oxo, an unsubstituted (C₁-C₁₈) aminoalkyloxy,an unsubstituted (C₁-C₁₈) aminoalkyloxy-(C₁-C₁₈) alkyl, an unsubstituted(C₁-C₁₈) aminoalkylcarboxy, an unsubstituted (C₁-C₁₈)aminoalkylaminocarbonyl, an unsubstituted (C₁-C₁₈)aminoalkylcarboxamido, an unsubstituted di(C₁-C₁₈ alkyl)aminoalkyl, anunsubstituted C-carboxy(C₁-C₁₈)alkyl, unsubstituted (C₁-C₁₈)guanidinoalkyloxy, unsubstituted (C₁-C₁₈) quaternary ammoniumalkylcarboxy, and unsubstituted (C₁-C₁₈) guanidinoalkyl carboxy.

In some embodiments, the compounds or pharmaceutically acceptable saltsthereof of Formula (V) can be also represented by Formula (I):

wherein fused rings A, B, C, and D are independently saturated or fullyor partially unsaturated; and each of R₁ through R₄, R₆, R₇, R₁₁, R₁₂,R₁₆, R₁₇, and R₁₈ is independently selected from the group consisting ofhydrogen, hydroxyl, a substituted or unsubstituted (C₁-C₁₀) alkyl,(C₁-C₁₀) hydroxyalkyl, (C₁-C₁₀) alkyloxy-(C₁-C₁₀) alkyl, (C₁-C₁₀)alkylcarboxy-(C₁-C₁₀) alkyl, C₁-C₁₀) alkylamino-(C₁-C₁₀) alkyl, (C₁-C₁₀)alkylamino-(C₁-C₁₀) alkylamino, (C₁-C₁₀) alkylamino-(C₁-C₁₀)alkylamino-(C₁-C₁₀) alkylamino, a substituted or unsubstituted (C₁-C₁₀)aminoalkyl, a substituted or unsubstituted aryl, a substituted orunsubstituted arylamino-(C₁-C₁₀) alkyl, (C₁-C₁₀) haloalkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, oxo, a linking group attached to a secondsteroid, a substituted or unsubstituted (C₁-C₁₀) aminoalkyloxy, asubstituted or unsubstituted (C₁-C₁₀) aminoalkyloxy-(C₁-C₁₀) alkyl, asubstituted or unsubstituted (C₁-C₁₀) aminoalkylcarboxy, a substitutedor unsubstituted (C₁-C₁₀) aminoalkylaminocarbonyl, a substituted orunsubstituted (C₁-C₁₀) aminoalkylcarboxamido, a substituted orunsubstituted C-carboxy(C₁-C₁₀)alkyl, H₂N—HC(Q₅)-C(O)—O—,H₂N—HC(Q₅)-C(O)—N(H)—, (C₁-C₁₀) azidoalkyloxy, (C₁-C₁₀) cyanoalkyloxy,P.G.-HN—HC(Q₅)-C(O)—O—, (C₁-C₁₀) guanidinoalkyloxy, (C₁-C₁₀) quaternaryammonium alkylcarboxy, and (C₁-C₁₀) guanidinoalkyl carboxy, where Q₅ isa side chain of any amino acid (including the side chain of glycine,i.e., H), PG. is an amino protecting group, and R₅, R₈, R₉, R₁₀, R₁₃,and R₁₄ is each independently: deleted when one of fused rings A, B, C,or D is unsaturated so as to complete the valency of the carbon atom atthat site, or selected from the group consisting of hydrogen, hydroxyl,10 a substituted or unsubstituted (C₁-C₁₀) alkyl, (C₁-C₁₀) hydroxyalkyl,(C₁-C₁₀) alkyloxy-(C₁-C₁₀) alkyl, a substituted or unsubstituted(C₁-C₁₀) aminoalkyl, a substituted or unsubstituted aryl, C₁-C₁₀haloalkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, oxo, a linking group attachedto a second steroid, a substituted or unsubstituted (C₁-C₁₀)aminoalkyloxy, a substituted or unsubstituted (C₁-C₁₀)aminoalkylcarboxy, a substituted or unsubstituted (C₁-C₁₀)aminoalkylaminocarbonyl, H₂N—HC(Q₅)-C(O)—O—, H₂N—HC(Q₅)-C(O)—N(H)—,(C₁-C₁₀) azidoalkyloxy, (C₁-C₁₀) cyanoalkyloxy, P.G.-HN—HC(Q₅)-C(O)—O—,(C₁-C₁₀) guanidinoalkyloxy, and (C₁-C₁₀) guanidinoalkylcarboxy, where Q₅is a side chain of any amino acid, PG. is an amino protecting group, andprovided that at least two of R₁ through R₁₄ are independently selectedfrom the group consisting of a substituted or unsubstituted (C₁-C₁₀)aminoalkyloxy, (C₁-C₁₀) alkylcarboxy-(C₁-C₁₀) alkyl, (C₁-C₁₀)alkylamino-(C₁-C₁₀) alkylamino, (C₁-C₁₀) alkylamino-(C₁-C₁₀)alkylamino-(C₁-C₁₀) alkylamino, a substituted or unsubstituted (C₁-C₁₀)aminoalkylcarboxy, a substituted or unsubstituted arylamino(C₁-C₁₀)alkyl, a substituted or unsubstituted (C₁-C₁₀) aminoalkyloxy-(C₁-C₁₀)alkyl, a substituted or unsubstituted (C₁-C₁₀) aminoalkylaminocarbonyl,(C₁-C₁₀) quaternary ammonium alkylcarboxy, H₂N—HC(Q₅)C(O)—O—,H₂N—HC(Q₅)-C(O)—N(H)—, (C₁-C₁₀) azidoalkyloxy, (C₁-C₁₀)cyanoalkyloxy,PG.-HN—HC(Q₅)-C(O)—O—, (C₁-C₁₀) guanidinoalkyloxy, and (C₁-C₁₀)guanidinoalkylcarboxy; or a pharmaceutically acceptable salt thereof.

In some embodiments, rings A, B, C, and D are independently saturated,heterocyclic, and/or non-heterocyclic.

In some embodiments, R₃, R₇, R₁₂, and R₁₈ are independently selectedfrom the group consisting of hydrogen, an unsubstituted (C₁-C₁₈) alkyl,unsubstituted (C₁-C₁₈) hydroxyalkyl, unsubstituted (C₁-C₁₈)alkyloxy-(C₁-C₁₈) alkyl, unsubstituted (C₁-C₁₈) alkylcarboxy-(C₁-C₁₈)alkyl, unsubstituted (C₁-C₁₈) alkylamino-(C₁-C₁₈)alkyl, unsubstituted(C₁-C₁₈) alkylamino-(C₁-C₁₈) alkylamino, unsubstituted (C₁-C₁₈)alkylamino-(C₁-C₁₈) alkylamino-(C₁-C₁₈) alkylamino, an unsubstituted(C₁-C₁₈) aminoalkyl, an unsubstituted arylamino-(C₁-C₁₈) alkyl, anunsubstituted (C₁-C₁₈) aminoalkyloxy, an unsubstituted (C₁-C₁₈)aminoalkyloxy-(C₁-C₁₈) alkyl, an unsubstituted (C₁-C₁₈)aminoalkylcarboxy, an unsubstituted (C₁-C₁₈) aminoalkylaminocarbonyl, anunsubstituted (C₁-C₁₈) aminoalkylcarboxamido, an unsubstituted di(C₁-C₁₈alkyl)aminoalkyl, an unsubstituted C-carboxy(C₁-C₁₈)alkyl, unsubstituted(C₁-C₁₈) guanidinoalkyloxy, unsubstituted (C₁-C₁₈) quaternary ammoniumalkylcarboxy, and unsubstituted (C₁-C₁₈) guanidinoalkyl carboxy; and R₁,R₂, R₄, R₅, R₆, R₈, R₉, R₁₀, R₁₁, R₁₃, R₁₄, R₁₅, R₁₆, and R₁₇ areindependently selected from the group consisting of hydrogen andunsubstituted (C₁-C₆) alkyl.

In some embodiments, R₁, R₂, R₄, R₅, R₆, R₈, R₁₀, R₁₁, R₁₄, R₁₆, and R₁₇are each hydrogen; and R₉ and R₁₃ are each methyl.

In some embodiments, R₃, R₇, R₁₂, and R₁₈ are independently selectedfrom the group consisting of aminoalkyloxy; aminoalkylcarboxy;alkylaminoalkyl; alkoxycarbonylalkyl; alkylcarbonylalkyl;di(alkyl)aminoalkyl; C-carboxyalkyl; alkoxycarbonylalkyl; andalkylcarboxyalkyl.

In some embodiments, R₃, R₇, and R₁₂ are independently selected from thegroup consisting of aminoalkyloxy and aminoalkylcarboxy; and R₁₈ isselected from the group consisting of alkylaminoalkyl;alkoxycarbonylalkyl; alkylcarbonyloxyalkyl; di(alkyl)aminoalkyl;alkylaminoalkyl; alkyoxycarbonylalkyl; and alkylcarboxyalkyl.

In some embodiments, R₃, R₇, and R₁₂ are the same substituent ordifferent substituents and/or may be independently an aminoalkyloxyand/or an aminoalkylcarboxy. In some embodiments, R₁₈ isalkylaminoalkyl, alkoxycarbonylalkyl, di(alkyl)aminoalkyl,C-carboxyalkyl, or an alkylcarboxyalkyl. In some embodiments, R₃, R₇,R₁₂, and R₁₈ are independently selected from the group consisting ofamino-C₃-alkyloxy; amino-C₃-alkyl-carboxy; C₈-alkylamino-C₅-alkyl;C₈-alkoxy-carbonyl-C₄-alkyl; C₈-alkyl-carbonyl-C₄-alkyl;di-(C₅-alkyl)amino-C₅-alkyl; C-carboxy-C₄-alkyl;C₁₃-alkylamino-C₅-alkyl; C₆-alkoxy-carbonyl-C₄-alkyl; andC₆-alkyl-carboxy-C₄-alkyl.

In some embodiments, the compounds or pharmaceutically acceptable saltsthereof of Formula (V) can be also represented by Formula (Ia):

In some embodiments, R₃, R₇, and R₁₂ are independently selected from thegroup consisting of hydrogen, an unsubstituted (C₁-C₂₂) alkyl,unsubstituted (C₁-C₂₂) hydroxyalkyl, unsubstituted (C₁-C₂₂)alkyloxy-(C₁-C₂₂) alkyl, unsubstituted (C₁-C₂₂) alkylcarboxy-(C₁-C₂₂)alkyl, unsubstituted (C₁-C₂₂) alkylamino-(C₁-C₂₂)alkyl, unsubstituted(C₁-C₂₂) alkylamino-(C₁-C₂₂) alkylamino, unsubstituted (C₁-C₂₂)alkylamino-(C₁-C₂₂) alkylamino-(C₁-C₁₈) alkylamino, an unsubstituted(C₁-C₂₂) aminoalkyl, an unsubstituted arylamino-(C₁-C₂₂) alkyl, anunsubstituted (C₁-C₂₂) aminoalkyloxy, an unsubstituted (C₁-C₂₂)aminoalkyloxy-(C₁-C₂₂) alkyl, an unsubstituted (C₁-C₂₂)aminoalkylcarboxy, an unsubstituted (C₁-C₂₂) aminoalkylaminocarbonyl, anunsubstituted (C₁-C₂₂) aminoalkylcarboxamido, an unsubstituted di(C₁-C₂₂alkyl)aminoalkyl, unsubstituted (C₁-C₂₂) guanidinoalkyloxy,unsubstituted (C₁-C₂₂) quaternary ammonium alkylcarboxy, andunsubstituted (C₁-C₂₂) guanidinoalkyl carboxy.

In some embodiments, R₃, R₇, and R₁₂ are independently selected from thegroup consisting of hydrogen, an unsubstituted (C₁-C₆) alkyl,unsubstituted (C₁-C₆) hydroxyalkyl, unsubstituted (C₁-C₁₆)alkyloxy-(C₁-C₅) alkyl, unsubstituted (C₁-C₁₆) alkylcarboxy-(C₁-C₅)alkyl, unsubstituted (C₁-C₁₆) alkylamino-(C₁-C₅)alkyl, unsubstituted(C₁-C₁₆) alkylamino-(C₁-C₅) alkylamino, unsubstituted (C₁-C₁₆)alkylamino-(C₁-C₁₆) alkylamino-(C₁-C₅) alkylamino, an unsubstituted(C₁-C₁₆) aminoalkyl, an unsubstituted arylamino-(C₁-C₅) alkyl, anunsubstituted (C₁-C₅) aminoalkyloxy, an unsubstituted (C₁-C₁₆)aminoalkyloxy-(C₁-C₅) alkyl, an unsubstituted (C₁-C₅) aminoalkylcarboxy,an unsubstituted (C₁-C₅) aminoalkylaminocarbonyl, an unsubstituted(C₁-C₅) aminoalkylcarboxamido, an unsubstituted di(C₁-C₅alkyl)amino-(C₁-C₅) alkyl, unsubstituted (C₁-C₅) guanidinoalkyloxy,unsubstituted (C₁-C₁₆) quaternary ammonium alkylcarboxy, andunsubstituted (C₁-C₁₆) guanidinoalkylcarboxy.

In some embodiments, R₃, R₇, and R₁₂ are independently selected from thegroup consisting of aminoalkyloxy; aminoalkylcarboxy; alkylaminoalkyl;alkoxycarbonylalkyl; alkylcarbonylalkyl; di(alkyl)aminoalkyl;alkylcarboxyalkyl; and hydroxyalkyl.

In some embodiments, R₃, R₇, and R₁₂ are independently selected from thegroup consisting of aminoalkyloxy and aminoalkylcarboxy.

In some embodiments, R₃, R₇, and R₁₂ are the same. In some embodiments,R₃, R₇, and R₁₂ are aminoalkyloxy. In some embodiments, R₃, R₇, and R₁₂are aminoalkylcarboxy.

In some embodiments, R₃, R₇, and R₁₂ are independently selected from thegroup consisting of amino-C₃-alkyloxy; amino-C₃-alkyl-carboxy;C₈-alkylamino-C₅-alkyl; C₈-alkoxy-carbonyl-C₄-alkyl;C₈-alkyl-carbonyl-C₄-alkyl; di-(C₅-alkyl)amino-C₅-alkyl;C₁₃-alkylamino-C₅-alkyl; C₆-alkoxy-carbonyl-C₄-alkyl;C₆-alkyl-carboxy-C₄-alkyl; and C₁₆-alkylamino-C₅-alkyl.

In some embodiments, CSA compounds as disclosed herein can be a compoundof Formula (I), Formula (II), Formula (III), or salts thereof wherein atleast R₁₈ of the steroidal backbone includes amide functionality inwhich the carbonyl group of the amide is positioned between the amidonitrogen of the amide and fused ring D of the steroidal backbone. Forexample, any of the embodiments described above can substitute R₁₈ foran R₁₈ including amide functionality in which the carbonyl group of theamide is positioned between the amido nitrogen of the amide and fusedring D of the steroidal backbone.

In some embodiments, at least R₁₈ can have the following structure:—R₂₀—(C═O)—N—R₂₁R₂₂wherein R₂₀ is omitted or alkyl, alkenyl, alkynyl, or aryl, and R₂₁ andR₂₂ are independently selected from the group consisting of hydrogen,alkyl, alkenyl, alkynyl, or aryl, provided that at least one of R₂₁ andR₂₂ is not hydrogen.

In some embodiments, R₂₁ and R₂₂ are independently selected from thegroup consisting of hydrogen, C₁-C₂₄ alkyl, C₂-C₂₄ alkenyl, C₂-C₂₄alkynyl, C₆ or C₁₀ aryl, 5 to 10 membered heteroaryl, 5 to 10 memberedheterocyclyl, C₇₋₁₃ aralkyl, (5 to 10 membered heteroaryl)-C₁-C₆ alkyl,C₃₋₁₀ carbocyclyl, C₄₋₁₀ (carbocyclyl)alkyl, (5 to 10 memberedheterocyclyl)-C₁-C₆ alkyl, amido, and a suitable amine protecting group,provided that at least one of R₂₁ and R₂₂ is not hydrogen. In someembodiments, R₂₁ and R₂₂, together with the atoms to which they areattached, form a 5 to 10 membered heterocyclyl ring.

In some embodiments, the compounds or pharmaceutically acceptable saltsthereof of Formula (Ia) may be one of the following or selected from thefollowing group:

The foregoing compounds may be provided as a pharmaceutically acceptablesalt such as, but not limited to, a hydrochloride salt or atri-hydrochloride salt.

In some embodiments, compounds comprise a ring system of at least 4fused rings, where each of the rings has from 5-7 atoms. The ring systemhas two faces, and contains 3 chains attached to the same face. Each ofthe chains contains a nitrogen-containing group that is separated fromthe ring system by at least one atom; the nitrogen-containing group isan amino group, e.g., a primary amino group, or a guanidino group.

The compound can also contain a hydrophobic group. In some embodiments,the hydrophobic group is a substituted (C₃₋₁₀) aminoalkyl group, a(C₁-C₁₀) alkyloxy (C₃₋₁₀) alkyl group, or a (C₁-C₁₀) alkylamino (C₃₋₁₀)alkyl group, attached to the steroid backbone. In some embodiments, thehydrophobic group is a substituted, branched, or unbranched substitutentwith greater than 12, 16, 18, 20, or 22 carbons. In some embodiments,the hydrophobic group may include a hydrocarbon chain of at least 9, 11,or 13 carbons distal to a heteroatom. In some embodiments, a compoundhaving a structure according to Formula (V) includes a hydrophobic groupat R¹⁸.

In some embodiments, the compounds set forth herein preserve certainstereochemical and electronic characteristics found in steroids. Theterm “same configuration” as used herein refers to substituents on thefused steroid having the same stereochemical orientation. For example,in some embodiments, substituents R₃, R₇ and R₁₂ are all β-substitutedor α-substituted.

In some embodiments, compounds include, but are not limited to,compounds having amine or guanidine groups covalently attached to asteroid backbone or scaffold at any carbon position, e.g., cholic acid.In various embodiments, a group is covalently attached at any one, ormore, of positions C₃, C₇ and C₁₂ of the steroid backbone or scaffold.In additional embodiments, a group is absent from anyone, or more, ofpositions C₃, C₇ and C₁₂ of the steroid backbone or scaffold. Compoundsthat include such groups can include a tether, the tether havingvariable chain length or size. As used herein, the terms “tether” or“tethered,” when used in reference to a compound, refers to the chain ofatoms between the steroid backbone or scaffold and a terminal amino orguanidine group. In various embodiments, a tether is covalently attachedat anyone, or more, of positions C₃, C₇ and C₁₂. In additionalembodiments, a tether is lacking at anyone, or more, of positions C₃, C₇and C₁₂. A tether length may include the heteroatom (O or N) covalentlyattached to the steroid backbone. The tether may include a hydrolysablelinkage such as an ester linkage.

In some embodiments, other ring systems can also be used, e.g., 5-memberfused rings. Compounds with backbones having a combination of 5- and6-membered rings are also contemplated. Amine or guanidine groups can beseparated from the backbone by at least one, two, three, four or moreatoms. The backbone can be used to orient the amine or guanidine groupson one face, or plane, of the steroid. For example, a scheme showing acompound having primary amino groups on one face, or plane, of abackbone is shown in Scheme I above.

The compounds and compositions disclosed herein are optionally preparedas pharmaceutically acceptable salts. The term “pharmaceuticallyacceptable salt” as used herein is a broad term, and is to be given itsordinary and customary meaning to a skilled artisan (and is not to belimited to a special or customized meaning), and refers withoutlimitation to a salt of a compound that does not cause significantirritation to an organism to which it is administered and does notabrogate the biological activity and properties of the compound. In someembodiments, the salt is an acid addition salt of the compound.Pharmaceutical salts can be obtained by reacting a compound withinorganic acids such as hydrohalic acid (e.g., hydrochloric acid orhydrobromic acid), sulfuric acid, nitric acid, and phosphoric acid.Pharmaceutical salts can also be obtained by reacting a compound with anorganic acid such as aliphatic or aromatic carboxylic or sulfonic acids,for example formic acid, acetic acid, propionic acid, glycolic acid,pyruvic acid, malonic acid, maleic acid, fumaric acid, trifluoroaceticacid, benzoic acid, cinnamic acid, mandelic acid, succinic acid, lacticacid, malic acid, tartaric acid, citric acid, ascorbic acid, nicotinicacid, methanesulfonic acid, ethanesulfonic acid, p-toluensulfonic acid,salicylic acid, stearic acid, muconic acid, butyric acid, phenylaceticacid, phenylbutyric acid, valproic acid, 1,2-ethanedisulfonic acid,2-hydroxyethanesulfonic acid, benzenesulfonic acid,2-naphthalenesulfonic acid, or naphthalenesulfonic acid. Pharmaceuticalsalts can also be obtained by reacting a compound with a base to form asalt such as an ammonium salt, an alkali metal salt, such as a lithium,sodium or a potassium salt, an alkaline earth metal salt, such as acalcium, magnesium or aluminum salt, a salt of organic bases such asdicyclohexylamine, N-methyl-D-glucamine, tris(hydroxymethyl)methylamine,C₁-C₇ alkylamine, cyclohexylamine, dicyclohexylamine, triethanolamine,ethylenediamine, ethanolamine, diethanolamine, triethanolamine,tromethamine, and salts with amino acids such as arginine and lysine; ora salt of an inorganic base, such as aluminum hydroxide, calciumhydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide, orthe like.

The forgoing compositions may be used for the novel use of increasingthe rate of wound healing in a subject with a tissue wound.

Tissue Treatment Compositions

While it is possible for the compounds described herein to beadministered alone, it may be preferable to formulate the compounds aspharmaceutical compositions suitable for application to the tissuewound. As such, in yet another aspect, tissue treatment compositionsuseful in the methods and uses of the disclosed embodiments areprovided. More particularly, the tissue treatment compositions describedherein may be useful, inter alia, for treating or promoting woundhealing in a subject. A tissue treatment composition is any compositionthat may be administered in vitro or in vivo or both to a subject inorder to promote or enhance wound healing. In a preferred embodiment, atissue treatment compositions may be administered in vivo.

As used herein the terms “pharmaceutically acceptable” and“physiologically acceptable” mean a biologically compatible formulation,gaseous, liquid or solid, or mixture thereof, which is suitable for oneor more routes of administration, in vivo delivery, or contact. Aformulation is compatible in that it does not destroy activity of anactive ingredient therein (e.g., a CSA), or induce adverse side effectsthat far outweigh any prophylactic or therapeutic effect or benefit.

In an embodiment, the tissue treatment compositions may be formulatedwith pharmaceutically acceptable excipients such as carriers, solvents,stabilizers, adjuvants, diluents, etc., depending upon the particularmode of administration and dosage form. The tissue treatmentcompositions should generally be formulated to achieve a physiologicallycompatible pH, and may range from a pH of about 3 to a pH of about 11,preferably about pH 3 to about pH 7, depending on the formulation androute of administration. In alternative embodiments, it may be preferredthat the pH is adjusted to a range from about pH 5.0 to about pH 8. Moreparticularly, the tissue treatment compositions may comprise atherapeutically or prophylactically effective amount of at least onecompound as described herein, together with one or more pharmaceuticallyacceptable excipients. Optionally, the tissue treatment compositions maycomprise a combination of the compounds described herein, or may includea second active ingredient useful in the treatment or prevention ofbacterial infection (e.g., anti-bacterial or anti-microbial agents).

Formulations, e.g., for parenteral or oral administration, are mosttypically solids, liquid solutions, emulsions or suspensions, whileinhalable formulations for pulmonary administration are generallyliquids or powders, with powder formulations being generally preferred.A preferred tissue treatment composition may also be formulated as alyophilized solid that is reconstituted with a physiologicallycompatible solvent prior to administration. Alternative tissue treatmentcompositions may be formulated as syrups, creams, ointments, tablets,and the like.

In general, formulations are prepared by uniformly and intimatelyassociating the active ingredient with liquid carriers or finely dividedsolid carriers or both, and then, if necessary, shaping the product. Forexample, a tablet may be made by compression or molding. Compressedtablets may be prepared by compressing, in a suitable machine, an activeingredient (e.g., a CSA) in a free-flowing form such as a powder orgranules, optionally mixed with a binder, lubricant, inert diluent,preservative, surface-active or dispersing agent. Molded tablets may beproduced by molding, in a suitable apparatus, a mixture of powderedcompound (e.g., CSA) moistened with an inert liquid diluent. The tabletsmay optionally be coated or scored and may be formulated so as toprovide a slow or controlled release of the active ingredient therein.

Cosolvents and adjuvants may be added to the formulation. Non-limitingexamples of cosolvents contain hydroxyl groups or other polar groups,for example, alcohols, such as isopropyl alcohol; glycols, such aspropylene glycol, polyethyleneglycol, polypropylene glycol, glycolether; glycerol; polyoxyethylene alcohols and polyoxyethylene fatty acidesters. Adjuvants include, for example, surfactants such as, soyalecithin and oleic acid; sorbitan esters such as sorbitan trioleate; andpolyvinylpyrrolidone.

Additionally, the tissue treatment compositions may be in the form of asterile injectable preparation, such as a sterile injectable aqueousemulsion or oleaginous suspension. This emulsion or suspension may beformulated according to the known art using those suitable dispersing orwetting agents and suspending agents, which have been mentioned above.The sterile injectable preparation may also be a sterile injectablesolution or suspension in a non-toxic parenterally acceptable diluent orsolvent, such as a solution in 1,2-propane-diol.

The sterile injectable preparation may also be prepared as a lyophilizedpowder. Among the acceptable vehicles and solvents that may be employedare water, Ringer's solution, and isotonic sodium chloride solution. Inaddition, sterile fixed oils may be employed as a solvent or suspendingmedium. For this purpose any bland fixed oil may be employed includingsynthetic mono- or diglycerides. In addition, fatty acids such as oleicacid may likewise be used in the preparation of injectables.

To obtain a stable water-soluble dose form of a tissue treatmentcompositions, a pharmaceutically acceptable salt of a compound describedherein may be dissolved in an aqueous solution of an organic orinorganic acid, such as 0.3 M solution of succinic acid, or morepreferably, citric acid. If a soluble salt form is not available, thecompound may be dissolved in a suitable co-solvent or combination ofco-solvents. Examples of suitable co-solvents include alcohol, propyleneglycol, polyethylene glycol 300, polysorbate 80, glycerin and the likein concentrations ranging from about 0 to about 60% of the total volume.In one embodiment, the active compound is dissolved in DMSO and dilutedwith water.

The pharmaceutical composition may also be in the form of a solution ofa salt form of the active ingredient in an appropriate aqueous vehicle,such as water or isotonic saline or dextrose solution. Also contemplatedare compounds which have been modified by substitutions or additions ofchemical or biochemical moieties which make them more suitable fordelivery (e.g., increase solubility, bioactivity, palatability, decreaseadverse reactions, etc.), for example by esterification, glycosylation,PEGylation, etc.

In one embodiment, the compounds described herein may be formulated fororal administration in a lipid-based formulation suitable for lowsolubility compounds. Lipid-based formulations can generally enhance theoral bioavailability of such compounds.

In some exemplary embodiments, a CSA comprises a multimer (e.g., adimer, trimer, tetramer, or higher order polymer). In some exemplaryembodiments, the CSAs can be incorporated into pharmaceuticalcompositions or formulations. Such compositions/formulations are usefulfor administration to a subject, in vivo or ex vivo. Tissue treatmentcompositions and formulations include carriers or excipients foradministration to a subject.

Such formulations include solvents (aqueous or non-aqueous), solutions(aqueous or non-aqueous), emulsions (e.g., oil-in-water orwater-in-oil), suspensions, syrups, elixirs, dispersion and suspensionmedia, coatings, isotonic and absorption promoting or delaying agents,compatible with pharmaceutical administration or in vivo contact ordelivery. Aqueous and non-aqueous solvents, solutions and suspensionsmay include suspending agents and thickening agents. Suchpharmaceutically acceptable carriers include tablets (coated oruncoated), capsules (hard or soft), microbeads, powder, granules andcrystals. Supplementary active compounds (e.g., preservatives,antibacterial, antiviral and antifungal agents) can also be incorporatedinto the compositions.

Cosolvents and adjuvants may be added to the formulation. Non-limitingexamples of cosolvents contain hydroxyl groups or other polar groups,for example, alcohols, such as isopropyl alcohol; glycols, such aspropylene glycol, polyethyleneglycol, polypropylene glycol, glycolether; glycerol; polyoxyethylene alcohols and polyoxyethylene fatty acidesters. Adjuvants include, for example, surfactants such as, soyalecithin and oleic acid; sorbitan esters such as sorbitan trioleate; andpolyvinylpyrrolidone.

Additionally, it may be desirable to include other therapeuticallybeneficial agents in the formulation. For example, the vehicles orcarriers may also include humectants or moisturizers to maintain adesired moisture level in the treated area. Other possibilities includedrugs such as anesthetics or antibiotics, which provide other desiredeffects. Again, the possibilities are unlimited and are left to thepractitioner.

A tissue treatment composition contains a total amount of the activeingredient(s) sufficient to achieve an intended therapeutic effect. Insome embodiments, the tissue treatment composition includes the CSA in aweight/weight concentration of at least 0.001%, 0.01%, 0.1%, 1.0%,and/or less than 80%, 50%, 25%, 15%, 10%, or 5%, or within a rangebetween any of the foregoing lower and upper endpoints. In someembodiments, the tissue treatment composition includes the CSA in aweight/weight concentration of about 0.04%. In other embodiments, thetissue treatment composition includes the CSA in a weight/weightconcentration of less than 0.1%. In some embodiments, the tissuetreatment composition includes the CSA in a weight/weight concentrationof between about 0.01% and about 2.0%.

Methods and Uses

Methods disclosed herein include identifying a subject with a tissuewound and administering a tissue treatment composition including a CSA,thereby increasing the rate of wound healing. In some embodiments, themethod includes (i) providing a tissue treatment composition asdescribed above; (ii) identifying a subject in need of acceleratedhealing of a tissue wound; and (iii) contacting the tissue wound withthe tissue treatment composition to increase the rate of healingthereof.

In some embodiments, the subject is an animal. In some embodiments, theanimal is a mammal. The mammal may be a human or primate in someembodiments. A mammal includes any mammal, such as by way ofnon-limiting example, cattle, pigs, sheep, goats, horses, camels,buffalo, cats, dogs, rats, mice, and humans. Preferably the subject is ahuman, horse, or dog. In some embodiments, the subject is a vertebrate.In other embodiments, the subject is a non-human animal.

The subject has a tissue wound in need of healing and/or acceleratedhealing. Unless specified, the term “wound” is used herein in itsgeneric sense, meaning that it encompasses all types of wounds andinjuries. The term “wound” encompasses burns, ulcers, lacerations,incisions, etc. “Wound” and “lesion” may be used interchangeably herein,and unless the context specifically dictates otherwise, no distinctionis intended. Lesions/wounds can be acute or chronic. Wounds can be fullthickness, i.e., penetrating all layers of skin, or partial thickness,i.e., penetrating less than all layers of skin. Examples of acute woundsinclude, but are not limited to, surgical wounds (i.e., incisions),penetrating wounds, avulsion injuries, crushing injuries, shearinginjuries, burn injuries, lacerations, and bite wounds. Examples ofchronic wounds include, but are not limited to, ulcers, such as arterialulcers, venous ulcers, pressure ulcers, and diabetic ulcers. Of course,acute wounds can become chronic wounds.

In some embodiments, the tissue wound can be a navel of a newbornsubject (i.e., a newborn with navel ill).

The tissue treatment composition can be applied to an open wound or aclosed wound. In some embodiments the composition is applied to an openwound for a period of sufficient time to cause closure of the wound. Thecomposition can be applied at least once or twice daily at least 2, 4,8, or 16 times and/or for over a period of at least 2, 4, 8, or 16 days.

The compositions disclosed herein can be administered to any wound,anywhere it is desirable to promote wound healing. The compositions arealso useful to reduce scarring after a wound is closed and/or healed.The compositions can be applied in any form of vehicle or carrier,including but not limited to, liquids, gels, lotions, creams, pastes,and ointments. The means of application will depend upon what form thecomposition takes: liquids can be sprayed or poured, for example; gels,lotions, creams, pastes, and ointments can be rubbed or massaged, forexample. These and other forms, and/or carriers/vehicles, forcomposition delivery, are described in publications such as Remington'sPharmaceutical Science, and other similar publications.

The delivery forms can be homogeneous, e.g., forms in which thecomposition is in solution, or heterogeneous, e.g., forms in which thecomposition is contained within liposomes or microspheres. The forms canproduce an immediate effect, and can alternatively, or additionally,produce an extended effect. For example, liposomes, or microspheres, orother similar means of providing an extended release of the composition,can be used to extend the period during which the composition is exposedto the lesion; non-encapsulated compositions can also be provided for animmediate effect.

The delivery forms can also take the form of devices, which can deliverthe composition to a lesion for a desired period of time. Devicesinclude, but are not limited to, bandages, surgical dressings, gauzes,adhesive strips, surgical staples, clips, hemostats, intrauterinedevices, sutures, trocars, catheters, tubes, collagen sponges, andimplants. Implants include, but are not limited to, pills, pellets,rods, wafers, discs, and tablets.

Devices according to the disclosure can be prepared according to knownmethods, and can include, or be made from, polymeric material. In someinstances, the polymeric material will be an absorbable material and inother instances, a non-absorbable material, or in other instances aresorbable material. Devices can include absorbable, non-absorbable,resorbable materials, and combinations thereof.

Absorbable materials can be synthetic materials and non-syntheticmaterials. Absorbable synthetic materials include, but are not limitedto, cellulosic polymers, glycolic acid polymers, methacrylate polymers,ethylene vinyl acetate polymers, ethylene vinyl alcohol copolymers,polycaptrolactam, polyacetate, copolymers of lactide and glycolide,polydioxanone, polyglactin, poliglecaprone, polyglyconate,polygluconate, and combinations thereof. Absorbable non-syntheticmaterials include, but are not limited to, catgut, cargile membrane,fascia lata, gelatin, collagen, and combinations thereof.

Nonabsorbable synthetic materials include, but are not limited tonylons, rayons, polyesters, polyolefins, and combinations thereof.Non-absorbable non-synthetic materials include, but are not limited to,silk, dermal silk, cotton, linen, and combinations thereof.

Combinations of the foregoing devices and carriers/vehicles are alsoenvisioned. For example, a CSA gel or ointment can be impregnated into abandage or wound dressing for delivery of the CSA to the desiredlocation. As another example, an implantable absorbable device can beloaded with a CSA solution and release the solution from the device overa period as desired.

It may be desirable to provide for other conditions in the practice ofthe present methods. For example, it may be desirable to ensure that thetarget region of the lesion is sufficiently oxygenated; generally, it issufficient that atmospheric oxygen be present. It also may be desirableto maintain a desired level of moisture and a particular temperature; insome embodiments, a warm, moist environment is desirable. While notrequired, it may also be desirable to establish or maintain a sterileenvironment.

In some embodiments, the composition may be incorporated into a medicaldevice coating.

One of ordinary skill in the art to which these exemplary embodimentsbelong will understand that the compositions may be administered innumerous ways. For example, administration may mean simply applying thecompositions to a wound directly. In some exemplary embodiments,administration may be enteral, parenteral, or topical. Other exemplaryroutes of administration for contact or in vivo delivery which acompound can optionally be formulated include inhalation, respiration,intubation, intrapulmonary instillation, oral (buccal, sublingual,mucosal), intrapulmonary, rectal, vaginal, intrauterine, intradermal,topical, dermal, parenteral (e.g., subcutaneous, intramuscular,intravenous, intradermal, intraocular, intratracheal and epidural),intranasal, intrathecal, intraarticular, intracavity, transdermal,iontophoretic, ophthalmic, optical (e.g., corneal), intraglandular,intraorgan, intralymphatic.

Dosage

Compounds (e.g., CSAs), including pharmaceutical formulations can bepackaged in unit dosage forms for ease of administration and uniformityof dosage. A “unit dosage form” as used herein refers to a physicallydiscrete unit suited as unitary dosages for the subject to be treated;each unit containing a predetermined quantity of compound optionally inassociation with a pharmaceutical carrier (excipient, diluent, vehicleor filling agent) which, when administered in one or more doses, iscalculated to produce a desired effect (e.g., prophylactic ortherapeutic effect or benefit). Unit dosage forms can contain a dailydose or unit, daily sub-dose, or an appropriate fraction thereof, of anadministered compound (e.g., CSA). Unit dosage forms also include, forexample, capsules, troches, cachets, lozenges, tablets, ampules andvials, which may include a composition in a freeze-dried or lyophilizedstate; a sterile liquid carrier, for example, can be added prior toadministration or delivery in vivo. Unit dosage forms additionallyinclude, for example, ampules and vials with liquid compositionsdisposed therein. Unit dosage forms further include compounds fortransdermal administration, such as “patches” that contact with theepidermis of the subject for an extended or brief period of time. Theindividual unit dosage forms can be included in multi-dose kits orcontainers. Pharmaceutical formulations can be packaged in single ormultiple unit dosage forms for ease of administration and uniformity ofdosage.

Compounds (e.g., CSAs) can be administered in accordance with themethods at any frequency as a single bolus or multiple dose e.g., one,two, three, four, five, or more times hourly, daily, weekly, monthly, orannually or between about 1 to 10 days, weeks, months, or for as long asappropriate. Exemplary frequencies are typically from 1-7 times, 1-5times, 1-3 times, 2-times or once, daily, weekly or monthly. Timing ofcontact, administration ex vivo or in vivo delivery can be dictated bythe infection, pathogenesis, symptom, pathology or adverse side effectto be treated. For example, an amount can be administered to the subjectsubstantially contemporaneously with, or within about 1-60 minutes orhours of the onset of a symptom or adverse side effect, pathogenesis, orvaccination.

Doses may vary depending upon whether the treatment is therapeutic orprophylactic, the onset, progression, severity, frequency, duration,probability of or susceptibility of the symptom, the type pathogenesisto which treatment is directed, clinical endpoint desired, previous,simultaneous or subsequent treatments, general health, age, gender orrace of the subject, bioavailability, potential adverse systemic,regional or local side effects, the presence of other disorders ordiseases in the subject, and other factors that will be appreciated bythe skilled artisan (e.g., medical or familial history). Dose amount,frequency or duration may be increased or reduced, as indicated by theclinical outcome desired, status of the infection, symptom or pathology,any adverse side effects of the treatment or therapy. The skilledartisan will appreciate the factors that may influence the dosage,frequency and timing required to provide an amount sufficient oreffective for providing a prophylactic or therapeutic effect or benefit.

In instances where animal and/or human dosages for compounds have beenestablished for at least some condition, those same dosages may be used,or dosages that are between about 0.1% and 500%, more preferably betweenabout 25% and 250% of the established animal and/or human dosage. Whereno animal and/or human dosage is established, as will be the case fornewly-discovered pharmaceutical compositions, a suitable animal and/orhuman dosage can be inferred from ED₅₀ or ID₅₀ values, or otherappropriate values derived from in vitro or in vivo studies, asqualified by toxicity studies and efficacy studies in animals.

In cases of administration of a pharmaceutically acceptable salt,dosages may be calculated as the free base. As will be understood bythose of skill in the art, in certain situations it may be necessary toadminister the compounds disclosed herein in amounts that exceed, oreven far exceed, the above-stated, preferred dosage range in order toeffectively and aggressively treat particularly aggressive diseases orconditions.

From preliminary studies, it found that toxicity for CSA compounds wasgreatly reduced at 5 μM and lower doses. An optimal standalone dose ofCSAs may be 10 μM or less at, on, or near the tissue wound, but smalleramounts may be used to minimize toxicity as needed.

Kits

Kits comprising the tissue treatment compositions and instructions forperforming such methods are also disclosed. The disclosure also provideskits including compounds (e.g., CSA), combination compositions andpharmaceutical compositions/formulations thereof, packaged into asuitable packaging material. In one embodiment, a kit includes packagingmaterial, a CSA, and instructions. In various aspects, the instructionsare for administering the CSA to enhance wound healing in a subject witha tissue wound. Where the tissue treatment composition is to be sprayed,the kit may include a spray container.

The term “packaging material” refers to a physical structure housing oneor more components of the kit. The packaging material can maintain thecomponents sterilely, and can be made of material commonly used for suchpurposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampules,vials, tubes, etc.). A kit can contain a plurality of components, e.g.,two or more compounds alone or in combination with a wound healing agentor treatment or drug, optionally sterile.

A kit optionally includes a label or insert including a description ofthe components (type, amounts, doses, etc.), instructions for use invitro, in vivo, or ex vivo, and any other components therein. Labels orinserts include “printed matter,” e.g., paper or cardboard, or separateor affixed to a component, a kit or packing material (e.g., a box), orattached to an ampule, tube or vial containing a kit component. Labelsor inserts can additionally include a computer readable medium, such asa disk (e.g., floppy diskette, hard disk, ZIP disk), optical disk suchas CD- or DVD-ROM/RAM, DVD, MP3, magnetic tape, or an electrical storagemedia such as RAM and ROM or hybrids of these such as magnetic/opticalstorage media, FLASH media or memory type cards.

EXAMPLES Example 1

A horse with a chronic non-healing oral cutaneous fistula was firsttreated with two prior art treatments with no success. The firsttreatment was with an aqueous spray of Vetericyn (0.007% hypochlorite)for a period of 7 days. The second unsuccessful treatment used FuracinSalve rubbed onto the open wound and applied daily for a period of 5days with no appreciable improvement in wound healing. The wound wassuccessfully treated in 7 days by spraying a composition of 0.04% CSA-44in water to the wound once daily for 7 days. The CSA-44 compound had thefollowing structure:

Closer of the fistula and healthy tissue were achieved in 7 days.

Example 2

A horse with a torn right front shoulder on a T post had a wound thatwas approximately 4 inches by 6 inches and 3 inches deep. This woundoccurs commonly in horses and usually takes 3 to 6 months to heal.Typically this wound will result in a significant loss of muscle in theregion. This wound was treated twice a day with a 0.04% solution ofCSA-44 in water. The tissue treatment composition was applied byspraying sufficient composition to wet the entire surface of the wound.The wound closed and completely healed in 28 days without any apparentmuscle loss of muscle. This result is surprising and unexpected giventhe location and size of wound and the typical length of time typicallyneed for this type of wound to heal.

Example 3

A reining broodmare in Texas was involved in an accident resulting in alaceration to her left hind cannon region. The wound was 3 inches inlength and 2 inches wide. The laceration extended all the way to thecannon bone, exposing the periosteum. This wound was also treated with a0.04% solution of CSA-44 in water twice daily. No wrap was applied(i.e., no immobilization). The wound closed in 11 days with normal hairgrowth at 30 days. This type of wound would usually require aggressivewraps for 3 months with the possibility of other procedures beingnecessary for the excessive granulation tissue that commonly develops.

Example 4

A horse with an extreme case of scratches was successfully healed usingCSA. Scratches is a fungal infection of the lower extremities in horses.This infection usually results in not only a fungal but also a secondarybacterial infection. In this case, the horse developed severe anddebilitating granulation tissue of the ankle resulting in poor mobilitydue to cracking and bleeding. Prior to treatment with CSA, the wound wasunsuccessfully treated with an oral antifungal, antibiotic (Naxcel), andVeterycin. The chronic infection persisted for over two years despitethe multiple attempts to treat with an antimicrobial. Due to thedebilitating wound the horse was slated for slaughter. However, thewound was successfully treated with a 0.04% of CSA-44 in water sprayedtwice daily for four weeks. Normal hair growth and tissue was present in4 weeks.

Example 5

A show mare developed an uncontrollable fungal infection of the skin.This malady is commonly referred to as summer itch. A 0.04% solution ofCSA-44 in water was used twice daily for 3 weeks resulting in completeregrowth of normal skin and hair. Normal treatment time periods for thisproblem usually range from 3 to 4 months.

Example 6

Genetically diabetic 8-week-old female mice (db/db, BKS.Cg-m+/+Leprdb/J)and heterozygous nondiabetic littermates (db/−) were purchased from TheJackson Laboratory (Bar Harbor, Me.). Strain and age of mouse werechosen because these mutant mice exhibit severe diabetic conditions,with hyperglycemia peaking between 8 and 12 weeks. They have also beenshown to have delayed wound healing with occlusive dressings. Mice wereordered to arrive at 7 weeks of age in order to decrease the chance oftravel stress effect as a confounding factor in our experiment. Micewere housed individually in the Veteran's Affairs Medical Center—SanFrancisco Animal Research Facility, maintained on a 12-hour light/darkcycle, and allowed ad libitum access to rodent chow and water. Mice wereanesthetized with an intraperitoneal injection of diluted chloralhydrate syrup (7½ gr/5 ml). Approximately 0.2 and 0.1 ml of a solutioncontaining 5 ml of syrup diluted with 7 ml of ddH₂0 was given to eachdb/db and db/− mouse respectively. For each mouse, the dorsal skin wasshaved, and cleansed with alcohol swabs. Mice were kept warm duringanesthesia and surgery by being huddled together until they regainedconsciousness. Two full-thickness 6-mm punch biopsy (Acuderm, Inc., Ft.Lauderdale, Fla.) wounds were created on the dorsal surface of the mice.Wounds were placed approximately 1-2 cm apart. Areas that were chosenwere free of anagen hair follicles. A piece of CSA impregnated film,approximately 6 mm in diameter, was placed in the wound. This film layadjacent, touching the wound edges. Mastisol was applied to the edges ofeach wound, which was then covered with a 1×1 cm Tegaderm semiocclusivedressing. To analyze the effect of the CSA impregnated film, digitalphotographs of wounds, including a metric ruler, were taken on day zeroand then every fourth day until complete wound closure was achieved.Prior to postoperative day 15, if Tegaderm dressing and/or impregnatedfilm fell off, they were promptly replaced with a new dressing and/orfilm. On postoperative day 15, Tegaderm dressings were removed andwounds were allowed to close by contraction. On subsequent days, scabswere gently removed from the wounds not covered because they decreasethe rate of wound contraction and impair assessment of wound closure.All wounds were compared to their original wound size using ImageJ(Rasband W S, NIH, http://rsb.info.nih.gov/ij/, 1997-2006).

Example 7

To determine the role of synthetic Ceragenins CSA-13, 44 and 90 in woundhealing using mesenchymal stem cells (MSC), targeted mRNA panels fromSABiosciences, and primary cells from Lonza were selected. Cells werepurchased from Lonza.com and used fresh for each test using recommendedmedia and culture conditions. After treatment, mRNA was isolated usingQiagen RNeasy Mini Kit®, and quantified using a NanoDrop 2000® by UV at260 nm and 260/280 ratio for purity. cDNA was made using a First StrandKit® from SABiosciences and processed for real time PCR using a kit fromthe same company for selected analysis of wound healing pathways.Results from q-PCR were uploaded to the SABiosciences site and toIngenuity.com web site for analysis and pathway mapping. SABioscienceswound healing array plates (Cat# PAHS-121) and innate/acquired immuneresponse plates (PAHS-052) were used. These arrays are fully validatedwhen used as recommended. On day 1, primary human MSC cells were platedat 200,000 cells/well using 6-well plates with 3 ml of recommendedmedia—hMSC Basal Medium+BulletKit (50 ml Growth Supplement, 10 mlL-Glutamine and 0.5 ml Gentamicin Sulfate Amphotercin-B) for 24 hours.Only early passages of cells were used, and never from frozen stock. Onday 2, cells were treated with compounds dissolved in DMSO diluted1:1000 or more to avoid effects of the solvent. Final testingconcentration for CSA-13 was 5.0 μM. Treatment lasted 8 hours, and wasfollowed by RNA isolation using QIAGEN RNeasy Mini Kit® (74104). RNA wasmeasured at 260/280 nm using a NanoDrop 2000® and normalized to 2.4 ngper well, cDNA preparation was done using QIAGEN First Strand kit330401. q-PCR was run as absolute quantification and threshold set at0.1 units. Dendritic cells were plated at 500,000 cells/well using24-well plate with 500 ul of Lonza LGM-3 Complete Growth Medium with andwithout compound. Treatment lasted 8 hours, and was followed by RNAisolation using QIAGEN RNeasy Mini Kit® (74104). RNA was measured at260/280 nm using NanoDrop2000® and normalized to 2.4 ng per well, cDNApreparation was done using QIAGEN First Strand kit 330401. PCR was runas absolute quantification and threshold set at 0.1 units. For the woundhealing array, strong upregulation of growth factors such as HB-EGF andcell migration factors such as MMP1 and CXCL2 were found, indicating aclear potential for CSA as a modulator of wound healing. Additional geneexpression data is provided in Tables 1-3 for CSA-13, 44, and 90,respectively.

TABLE 1 Gene Expression Results for CSA-13 Gene Symbol Fold RegulationCCL7 1.6632 CXCL1 1.6181 CXCL2 4.873 CXCL5 2.0582 F13A1 2.0916 FGF103.8659 HBEGF 3.255 IL2 1.865 IL6 3.1692 ITGA2 3.5659 MMP1 4.4172 PLAU1.7849 PLAUR 1.6286 PTGS2 3.3333 VEGFA 1.7274 VTN 2.0612 ANGPT1 −2.0046CSF2 −2.4867 F3 −3.3945 FGF2 −1.633 IL10 −1.6166 IL4 −1.9944 ITGB3−1.5243 PLAT −2.1487

TABLE 2 Gene Expression Results for CSA-44 Gene Symbol Fold RegulationCCL7 2.1961 COL1A2 1.5483 COL3A1 1.7385 CTSK 1.6388 CTSL2 1.7924 CXCL214.3964 EGFR 1.5364 F13A1 2.0963 FGF10 2.2811 FGF7 4.84 HBEGF 3.5463 HGF3.1098 IGF1 1.6877 IL2 2.1928 IL6 4.0387 ITGA2 16.0648 ITGB6 1.6323 MMP168.9688 MMP9 1.5543 PLAU 1.6131 PLAUR 2.5454 PTGS2 48.6907 TIMP1 1.6126VEGFA 4.6052 ACTA2 −1.9377 ANGPT1 −2.0857 CCL2 −3.1925 CDH1 −2.7158COL4A3 −2.4845 CSF2 −1.8551 CTGF −24.5295 FGF2 −1.6016 IL10 −2.0128ITGB3 −1.5802 PLAT −1.754 SERPINE1 −2.9618 TGFBR3 −2.0462 WISP1 −1.9722ACTB −1.7981

TABLE 3 Gene Expression Results for CSA-90 Gene Symbol Fold RegulationCCL7 2.2874 CTSK 1.5366 CTSL2 1.6306 CXCL1 3.1083 CXCL2 36.5878 EGFR1.6212 F13A1 2.1032 FGF10 1.9842 FGF7 5.1689 HBEGF 3.5988 HGF 2.3334IFNG 1.839 IGF1 2.2724 IL2 1.8522 IL6 7.2299 ITGA2 14.3637 ITGB6 1.9381MMP1 45.3753 MMP9 2.5652 PLAUR 1.9924 PTGS2 66.6189 TIMP1 1.8212 VEGFA3.3257 ACTA2 −2.5014 ANGPT1 −1.915 CCL2 −1.5793 COL4A3 −1.6195 CTGF−15.7171 IL10 −1.7382 ITGA3 −1.5127 ITGB3 −1.538 PLAT −2.14 SERPINE1−3.7307 TGFBR3 −1.5938 WISP1 −2.3543 ACTB −2.0745

Example 8

Infections acquired after burn injuries in combat soldiers increasechances of sepsis and death. These burn wounds are susceptible tobiofilm formation, an environment that can decrease treatmenteffectiveness. Most antimicrobial treatments do not target biofilms norpromote wound repair of infected wounds. Because of the lack of effectsassociated with current treatments, there is a need for antimicrobialagents that both promote wound healing processes in skin and eradicatepolymicrobial biofilms. Our body produces antimicrobial peptides as partof the innate defense against infections. Peptides, while effective invitro, often face degradation in the body. We hypothesize that peptideswith stability modifications and peptide-mimics maintain anti-biofilmpotency and induce wound healing pathways.

Standard microdilution methods were used to determine minimum inhibitoryconcentration (MIC) and minimum bactericidal concentration (MBC) of testagents (peptides and peptide-mimics) against drug resistantStaphylococcus. aureus (TCH1516) and Pseudomonas. aeruginosa (PAO1).Mixed-species biofilms of S. aureus and P. aeruginosa, grown incollagen-coated plates for 24 hours were tested for their susceptibilityto treatments as revealed by live/dead staining. Cytotoxicity of testagents to human immortalized keratinocytes (HaCaTs) was assessed using3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT).Promotion of HaCaT migration by test agents was tested using electriccell-substrate impedance sensing (ECIS®), which induced 2D woundhealing. The ability of test agents to stimulate angiogenesis wasevaluated using matrigel-induced tube formation of human veinendothelial cells (HUVECs).

LL-37, a human peptide and positive control, showed anti-biofilmactivity and induced wound healing. Ceragenins, a group ofpeptide-mimics, demonstrated stronger antibacterial and anti-biofilmactivities, but also showed increased toxicity to HaCaTs. Dermaseptin S1(DRS1), a frog skin peptide, increased in anti-biofilm activity whenshortened to the first 15 amino acids; replacing specific residues inthis sequence also affected its antibacterial and anti-biofilm activity.Preliminary studies show DRS1 peptides and ceragenins induced woundhealing and angiogenesis. Through high-throughput assessment, moleculesthat maximize anti-biofilm potential while improving wound healingfunctions were down-selected for further mechanistic investigation.

Furthermore, although the foregoing has been described in some detail byway of illustrations and examples for purposes of clarity andunderstanding, it will be understood by those of skill in the art thatnumerous and various modifications can be made without departing fromthe spirit of the present disclosure. Therefore, it should be clearlyunderstood that the forms disclosed herein are illustrative only and arenot intended to limit the scope of the present disclosure, but rather toalso cover all modification and alternatives coming with the true scopeand spirit of the invention.

What is claimed is:
 1. A method for increasing the rate of healing of a tissue wound, comprising: identifying a subject in need of accelerated healing of a tissue wound; and contacting the tissue wound with an effective amount of a tissue treatment composition to increase the rate of healing thereof, the tissue treatment composition comprising a cationic steroidal anti-microbial (CSA) compound of Formula V, or a pharmaceutically acceptable salt thereof, that promotes would healing and comprises a steroidal group and a plurality of cationic groups attached thereto:

where, rings A, B, C, and D are independently saturated, or are fully or partially unsaturated, provided that at least two of rings A, B, C, and D are saturated; m, n, p, and q are independently 0 or 1; R₁, R₂, R₄, R₅, R₆, R₈, R₉, R₁₀, R₁₁, R₁₃, R₁₄, R₁₅, R₁₆, and R₁₇ are independently selected from the group consisting of hydrogen and substituted or unsubstituted (C₁-C₆) alkyl; and R₃, R₇, and R₁₂ are independently selected from the group consisting of hydrogen, hydroxyl, substituted or unsubstituted (C₁-C₁₈) alkyl, substituted or unsubstituted (C₁-C₁₈) hydroxyalkyl, substituted or unsubstituted (C₁-C₁₈) alkyloxy-(C₁-C₁₈) alkyl, substituted or unsubstituted (C₁-C₁₈) alkylcarboxy-(C₁-C₁₈) alkyl, substituted or unsubstituted (C₁-C₁₈) alkylamino-(C₁-C₁₈) alkyl, substituted or unsubstituted (C₁-C₁₈) alkylamino-(C₁-C₁₈) alkylamino, substituted or unsubstituted (C₁-C₁₈) alkylamino-(C₁-C₁₈) alkylamino-(C₁-C₁₈) alkylamino, substituted or unsubstituted (C₁-C₁₈) aminoalkyl, substituted or unsubstituted aryl, substituted or unsubstituted arylamino-(C₁-C₁₈) alkyl, substituted or unsubstituted (C₁-C₁₈) haloalkyl, substituted or unsubstituted (C₂-C₆) alkenyl, substituted or unsubstituted (C₂-C₆) alkynyl, oxo, linking group attached to a second CSA, substituted or unsubstituted (C₁-C₁₈) aminoalkyloxy, substituted or unsubstituted (C₁-C₁₈) aminoalkyloxy-(C₁-C₁₈) alkyl, substituted or unsubstituted (C₁-C₁₈) aminoalkylcarboxy, substituted or unsubstituted (C₁-C₁₈) aminoalkylaminocarbonyl, substituted or unsubstituted (C₁-C₁₈) aminoalkylcarboxamido, substituted or unsubstituted di(C₁-C₁₈ alkyl) aminoalkyl, substituted or unsubstituted C-carboxy-(C₁-C₁₈) alkyl, H₂N—HC(Q₅)-C(O)—O—, H₂N—HC(Q₅)-C(O)—N(H)—, substituted or unsubstituted (C₁-C₁₈) azidoalkyloxy, substituted or unsubstituted (C₁-C₁₈) cyanoalkyloxy, P.G.-HN—HC(Q₅)-C(O)—O, substituted or unsubstituted (C₁-C₁₈) guanidinoalkyloxy, substituted or unsubstituted (C₁-C₁₈) quaternary ammonium alkyl carboxy, and substituted or unsubstituted (C₁-C₁₈) guanidinoalkylcarboxy, where Q₅ is a side chain of an amino acid and P.G. is an amino protecting group, provided that at least two of R₃, R₇, and R₁₂ are not hydrogen; and R₁₈ is selected from the group consisting of hydroxyl, substituted or unsubstituted (C₁-C₁₈) alkyl, substituted or unsubstituted (C₁-C₁₈) hydroxyalkyl, substituted or unsubstituted (C₁-C₁₈) alkyloxy-(C₁-C₁₈) alkyl, substituted or unsubstituted (C₁-C₁₈) alkylcarboxy-(C₁-C₁₈) alkyl, substituted or unsubstituted (C₁-C₁₈) alkylamino-(C₁-C₁₈) alkyl, substituted or unsubstituted (C₁-C₁₈) alkylamino-(C₁-C₁₈) alkylamino, substituted or unsubstituted amido, substituted or unsubstituted (C₁-C₁₈) aminoalkyl, substituted or unsubstituted aryl, substituted or unsubstituted arylamino-(C₁-C₁₈) alkyl, substituted or unsubstituted (C₁-C₁₈) haloalkyl, substituted or unsubstituted (C₂-C₆) alkenyl, substituted or unsubstituted (C₂-C₆) alkynyl, linking group attached to a second CSA, substituted or unsubstituted (C₁-C₁₈) aminoalkyloxy, substituted or unsubstituted (C₁-C₁₈) aminoalkyloxy-(C₁-C₁₈) alkyl, substituted or unsubstituted (C₁-C₁₈) aminoalkylcarboxy (C₁-C₁₈) alkyl, substituted or unsubstituted (C₁-C₁₈) aminoalkylaminocarbonyl, substituted or unsubstituted (C₁-C₁₈) aminoalkylcarboxamido, substituted or unsubstituted di(C₁-C₁₈ alkyl) aminoalkyl, substituted or unsubstituted C-carboxy-(C₁-C₁₈) alkyl, H₂N—HC(Q₅)-C(O)—O—, H₂N—HC(Q₅)-C(O)—N(H)—, substituted or unsubstituted (C₁-C₁₈) azidoalkyloxy, substituted or unsubstituted (C₁-C₁₈) cyanoalkyloxy, P.G.-HN—HC(Q₅)-C(O)—O, substituted or unsubstituted (C₁-C₁₈) guanidinoalkyloxy, substituted or unsubstituted (C₁-C₁₈) quaternary ammonium alkylcarboxy, and substituted or unsubstituted (C₁-C₁₈) guanidinoalkylcarboxy, where Q₅ is a side chain of an amino acid and P.G. is an amino protecting group.
 2. The method of claim 1, wherein R₁₈ has the following structure: —R₂₀—(C═O)—N—R₂₁R₂₂ wherein, R₂₀ is a substituted or unsubstituted alkyl, a substituted or unsubstituted alkenyl, a substituted or unsubstituted alkynyl, or a substituted or unsubstituted aryl; and R₂₁ and R₂₂ are independently selected from the group consisting of hydrogen, a substituted or unsubstituted C₁-C₂₄ alkyl, a substituted or unsubstituted C₂-C₂₄ alkenyl, a substituted or unsubstituted C₂-C₂₄ alkynyl, or a substituted or unsubstituted C₆ or C₁₀ aryl, 5 to 10 membered heteroaryl, 5 to 10 membered heterocyclyl, C₇₋₁₃ aralkyl, (5 to 10 membered heteroaryl)-C₁-C₆ alkyl, C₃₋₁₀ carbocyclyl, C₄₋₁₀ (carbocyclyl)alkyl, (5 to 10 membered heterocyclyl)-C₁-C₆ alkyl, amido, and an amine protecting group, provided that at least one of R₂₁ and R₂₂ is not hydrogen.
 3. The method of claim 1, wherein the tissue treatment composition is applied to an open wound for a period of time sufficient to cause closure of the wound.
 4. The method of claim 1, wherein the tissue treatment composition is applied to a closed wound.
 5. The method of claim 1, wherein the tissue treatment composition is applied to a tissue laceration or an incision site.
 6. The method of claim 1, wherein the tissue treatment composition is applied to the tissue wound in at least four separate applications and/or over a period of at least 4 days.
 7. The method of claim 1, wherein the tissue treatment composition is applied to the tissue wound by spraying.
 8. The method of claim 1, wherein the CSA compound is administered from a pharmaceutically acceptable device selected from the group consisting of bandages, surgical dressings, gauzes, adhesive strips, surgical staples, clips, hemostats, intrauterine devices, sutures, trocars, catheters, tubes, and implants.
 9. The method of claim 1, wherein the subject is a vertebrate.
 10. The method of claim 1, wherein the subject is mammal.
 11. The method of claim 10, wherein the subject is a non-human mammal.
 12. The method of claim 10, wherein the subject is a human, horse, or dog.
 13. The method of claim 10, wherein the subject is a newborn and the tissue wound is a navel of the newborn.
 14. The method of any claim 1, wherein the tissue treatment composition is for veterinary use.
 15. The method of claim 1, wherein the tissue wound is a burn.
 16. The method of claim 1, wherein the tissue wound is not a burn.
 17. The method of claim 1, wherein the tissue wound includes a chronic infection.
 18. The method of claim 1, wherein the tissue treatment composition includes the CSA in a concentration in a range between 0.01-5% wt/wt.
 19. The method of claim 1, wherein the tissue treatment composition includes the CSA in a concentration in a range between about 0.01-2.0% wt/wt.
 20. The method of claim 1, wherein the plurality of cationic groups are each attached to the steroidal group through a hydrolysable ester linkage.
 21. The method of claim 1, wherein the CSA compound has the Formula (Ia).


22. The method of claim 21, wherein at least R₃, R₇, and R₁₂ of Formula Ia are independently an aminoalkyloxy or an aminoalkylcarboxy.
 23. The method of claim 21, wherein R₁₈ of Formula Ia is selected from the group consisting of alkylaminoalkyl, di(alkyl)aminoalkyl, alkoxycarbonylalkyl, alkylcarboxyalkyl, C-carboxyalkyl, and alkylcarbonyloxyalkyl.
 24. The method of claim 21, wherein R₁₈ has the following structure: —R₂₀—(C═O)—N—R₂₁R₂₂ wherein, R₂₀ is a substituted or unsubstituted alkyl, a substituted or unsubstituted alkenyl, a substituted or unsubstituted alkynyl, or a substituted or unsubstituted aryl; and R₂₁ and R₂₂ are independently selected from the group consisting of hydrogen, a substituted or unsubstituted C₁-C₂₄ alkyl, a substituted or unsubstituted C₂-C₂₄ alkenyl, a substituted or unsubstituted C₂-C₂₄ alkynyl, or a substituted or unsubstituted C₆ or C₁₀ aryl, 5 to 10 membered heteroaryl, 5 to 10 membered heterocyclyl, C₇₋₁₃ aralkyl, (5 to 10 membered heteroaryl)-C₁-C₆ alkyl, C₃₋₁₀ carbocyclyl, C₄₋₁₀ (carbocyclyl)alkyl, (5 to 10 membered heterocyclyl)-C₁-C₆ alkyl, amido, and an amine protecting group, provided that at least one of R₂₁ and R₂₂ is not hydrogen.
 25. The method of claim 1, wherein the CSA compound, or a pharmaceutically acceptable salt thereof, is selected from the group consisting of:

pharmaceutically acceptable salts thereof.
 26. The method of claim 1, wherein the CSA compound, or a pharmaceutically acceptable salt thereof, is:

or a pharmaceutically acceptable salt thereof.
 27. A method of increasing the rate of healing of a tissue wound, comprising: identifying a subject in need of accelerated healing of a tissue wound; and contacting the tissue wound with an effective amount of a tissue treatment composition to increase the rate of healing thereof, the tissue treatment composition comprising a cationic steroidal anti-microbial (CSA) compound of Formula Ia or pharmaceutically acceptable salt thereof that promotes wound healing:

wherein, R₃, R₇, and R₁₂ are independently selected from the group consisting of independently selected from the group consisting of hydrogen, an unsubstituted (C₁-C₂₂) alkyl, unsubstituted (C₁-C₂₂) hydroxyalkyl, unsubstituted (C₁-C₂₂) alkyloxy-(C₁-C₂₂) alkyl, unsubstituted (C₁-C₂₂) alkylcarboxy-(C₁-C₂₂) alkyl, unsubstituted (C₁-C₂₂) alkylamino-(C₁-C₂₂)alkyl, unsubstituted (C₁-C₂₂) alkylamino-(C₁-C₂₂) alkylamino, unsubstituted (C₁-C₂₂) alkylamino-(C₁-C₂₂) alkylamino-(C₁-C₁₈) alkylamino, an unsubstituted (C₁-C₂₂) aminoalkyl, an unsubstituted arylamino-(C₁-C₂₇) alkyl, an unsubstituted (C₁-C₂₂) aminoalkyloxy, an unsubstituted (C₁-C₂₂) aminoalkyloxy-(C₁-C₂₂) alkyl, an unsubstituted (C₁-C₂₂) aminoalkylcarboxy, an unsubstituted (C₁-C₂₂) aminoalkyl-aminocarbonyl, an unsubstituted (C₁-C₂₂) aminoalkylcarboxamido, an unsubstituted di(C₁-C₂₂ alkyl)aminoalkyl, unsubstituted (C₁-C₂₂) guanidinoalkyloxy, unsubstituted (C₁-C₂₂) quaternary ammonium alkylcarboxy, and unsubstituted (C₁-C₂₂) guanidinoalkyl carboxy, provided that at least two of R₃, R₇, and R₁₂ are not hydrogen; and R₁₈ is selected from the group consisting of substituted or unsubstituted (C₁-C₁₈) alkyl carboxy-(C₁-C₁₈) alkyl, substituted or unsubstituted (C₁-C₁₈) alkylamino-(C₁-C₁₈) alkyl, substituted or unsubstituted amido, substituted or unsubstituted (C₁-C₁₈) aminoalkyl, linking group attached to a second CSA, substituted or unsubstituted (C₁-C₁₈) aminoalkyloxy, substituted or unsubstituted (C₁-C₁₈) aminoalkyloxy-(C₁-C₁₈) alkyl, substituted or unsubstituted (C₁-C₁₈) aminoalkylcarboxy (C₁-C₁₈) alkyl, and substituted or unsubstituted is alkyl) aminoalkyl.
 28. The method of claim 27, wherein Rig has the following structure: —R₂₀—(C═O)—N—R₂₁R₂₂ wherein, R₂₀ is omitted or a substituted or unsubstituted alkyl, alkenyl, alkynyl, or aryl; and R₂₁ and R₂₂ are independently selected from the group consisting of hydrogen, a substituted or unsubstituted alkyl, a substituted or unsubstituted alkenyl, a substituted or unsubstituted alkynyl, or a substituted or unsubstituted aryl, provided that at least one of R₂₁ and R₂₂ is not hydrogen.
 29. A method of increasing the rate of healing of a tissue wound, comprising: identifying a subject in need of accelerated healing of a tissue wound; and contacting the tissue wound with an effective amount of a cationic steroidal anti-microbial (CSA) compound, or a pharmaceutically acceptable salt thereof, selected from the group consisting of:

pharmaceutically acceptable salts thereof. 